Compositions and methods for increasing muscle growth

ABSTRACT

This disclosure is in the field of anti-Activin receptor IIB (ActRIIB) antibodies. In particular, it relates to the use of said antibodies for treating muscle disorders, such as muscle wasting due to disease or disuse.

TECHNICAL FIELD

This disclosure is in the field of anti-Activin receptor IIB (ActRIIB)antibodies. In particular, it relates to the use of said antibodies fortreating muscle disorders, such as muscle wasting due to disease ordisuse.

BACKGROUND ART

Activins are dimeric growth and differentiation factors which belong tothe transforming growth factor-beta (TGF-beta) superfamily ofstructurally related signaling proteins. Activins signal through aheterodimeric complex of receptor serine kinases which include at leasttwo type I (I and IB) and two type II (II and IIB, aka ACVR2A andACVR2B) receptors. These receptors are all transmembrane proteins,composed of a ligand-binding extracellular domain with cysteine-richregion, a transmembrane domain, and a cytoplasmic domain with predictedserine/threonine specificity. Type I receptors are essential forsignalling while type II receptors are required for binding ligands andfor expression of type I receptors. Type I and II receptors form astable complex after ligand binding resulting in the phosphorylation oftype I receptors by type II receptors.

The activin receptor II B (ActRIIB) is a receptor for myostatin. Theinteraction between myostatin and this receptor regulates the inhibitionof skeletal muscle differentiation via the Smad-dependent pathway. Thus,by inhibiting or preventing myostatin from binding to ActRIIB, one caninduce the formation of skeletal muscle.

Various groups have looked into this. Bogdanovich et al (Nature, 2002,420:418-421) describes that anti-myostatin antibodies were able to blockmyostatin, resulting in an increase in muscle mass in a mouse model ofDuchenne muscular dystrophy. Bradley et al (Cell Mol. Life Sci. 2008,65:2119-2124) have reviewed the different available approaches formodulating the myostatin/ActRIIB interaction, including theaforementioned anti-myostatin antibodies, inhibiting the release ofmature myostatin by administering the myostatin propeptide,administering follistatin to block the myostatin receptor, administeringHDAC inhibitors to induce follistatin production, administering analtered myostatin peptide which prevents myostatin from binding thereceptor and administering a soluble decoy receptor for myostatin.

Despite these potential therapies, there is no product available for thetreatment of patients. Indeed, recently one company cancelled itsanti-myostatin antibody project.

There is therefore a need for a method of increasing muscle mass andstrength in a patient.

DISCLOSURE OF THE INVENTION

It has been discovered that antibodies directed to the ActRIIB receptorcan prevent myostatin from binding to the receptor, thus preventing theinhibition of muscle differentiation by the Smad-dependent pathway. Thisleads to an increase in muscle mass and strength in a patient.

Therefore, in one aspect, the disclosure provides an anti-ActRIIBantibody, or a functional fragment thereof or functional proteincomprising an antigen-binding portion of an anti-ActRIIB antibody. Inone embodiment, the ActRIIB is human ActRIIB. The polypeptide sequenceof human ActRIIB is recited in SEQ ID NO: 181 (AAC64515.1, GI:3769443).In one embodiment, the antibody or functional protein is from a mammal,having an origin such as human or camelid. Thus the antibody may be achimeric, human or a humanized antibody. In a particular embodiment, theanti-ActRIIB antibody is characterized as having antigen-binding regionthat is specific for the target protein ActRIIB and binds to ActRIIB ora fragment of ActRIIB. In one embodiment, the antibody is suitable foruse in therapy.

In one embodiment, the antibodies according to the disclosure areActRIIB antagonists with no or low agonistic activity. In anotherembodiment, the antibody or functional fragment comprising anantigen-binding portion binds the target protein ActRIIB and decreasesthe binding of myostatin to ActRIIB to a basal level. In one aspect ofthis embodiment, the antibody or functional fragment reduces the amountof myostatin that binds to ActRIIB. In a further aspect of thisembodiment, the antibody or functional fragment completely preventsmyostatin from binding to ActRIIB. In a further embodiment, the antibodyor functional fragment inhibits Smad activation. In a furtherembodiment, the antibody or functional fragment inhibits activinreceptor type IIB mediated myostatin-induced inhibition of skeletaldifferentiation via the Smad-dependent pathway.

The binding may be determined by one or more assays that can be used tomeasure an activity which is either antagonism or agonism by theantibody. In one embodiment, the assays measure at least one of theeffects of the antibody on ActRIIB that include: inhibition of myostatinbinding to ActRIIB by ELISA, inhibition of myostatin induced signalling(for instance by a Smad dependent reporter gene assay), inhibition ofmyostatin induced Smad phosphorylation (P-Smad ELISA) and inhibition ofmyostatin induced inhibition of skeletal muscle cell differentiation(for instance by a creatine kinase assay).

In one embodiment, the disclosure provides antibodies that specificallybind to the myostatin binding region (i.e. ligand binding domain) ofActRIIB. This ligand binding domain consists of amino acids 19-134 ofSEQ ID NO: 181 and has been assigned SEQ ID NO: 182 herein.

In one embodiment, the antibodies bind to ActRIIB with a K_(D) of 100 nMor less, 10 nM or less, 1 nM or less. In one embodiment, the antibodiesof the disclosure bind to ActRIIB with an affinity of 100 pM or less(i.e. 100 pM, 50 pM, 10 pM, 1 pM or less). In one embodiment, theantibodies of the disclosure bind to ActRIIB with an affinity of between10 and 20 pM.

In one embodiment, the antibodies of the disclosure do not cross-reactwith an ActRIIB related protein, and more particularly do notcross-react with human ActRIIA (NP_001607.1, GI:4501897).

In one embodiment, the antibodies of the disclosure in one embodimentbind to ActRIIB rather than ActRIIA. In one embodiment, the antibodiesof the disclosure bind to ActRIIB with 5-fold greater affinity than theybind to ActRIIA, more particularly 10-fold, still more particularly50-fold, still more particularly 100-fold.

In one embodiment, the antibodies of the disclosure bind to ActRIIA withan affinity of 100 pM or more (i.e. 250 pM, 500 pM, 1 nM, 5 nM or more).

In one embodiment the antibodies of the disclosure are of the IgG2isotype.

In another embodiment, the antibodies of the disclosure are of the IgG1isotype. In a further embodiment, the antibodies of the disclosure areof the IgG1 isotype and have an altered effector function throughmutation of the Fc region. In one embodiment, said altered effectorfunction is reduced ADCC and CDC activity. In one embodiment, saidaltered effector function is silenced ADCC and CDC activity.

In another related embodiment, the antibodies according to thedisclosure are fully human or humanized IgG1 antibodies with no antibodydependent cellular cytotoxicity (ADCC) activity or CDC activity and bindto a region of ActRIIB consisting of amino acids 19-134 of SEQ IDNO:181.

In another related embodiment, the antibodies according to thedisclosure are fully human or humanized IgG1 antibodies with reducedantibody dependent cellular cytotoxicity (ADCC) activity or CDC activityand bind to a region of ActRIIB consisting of amino acids 19-134 of SEQID NO:181.

The present disclosure relates to isolated antibodies, particularlyhuman or humanized antibodies that inhibit myostatin binding to ActRIIBand activate skeletal muscle differentiation in vitro and in vivo. Incertain embodiments, the antibodies of the disclosure are derived fromparticular heavy and light chain sequences and/or comprise particularstructural features such as CDR regions comprising particular amino acidsequences. The disclosure provides isolated antibodies, methods ofmaking such antibodies, immunoconjugates and multivalent ormulti-specific molecules comprising such antibodies and pharmaceuticalcompositions containing the antibodies, immunoconjugates or bi-specificmolecules of the disclosure. The disclosure also relates to methods ofusing the antibodies to inhibit, i.e. antagonize, function of ActRIIB inorder to inhibit Smad activation and thereby induce skeletal muscledifferentiation, for example, resulting in the treatment of apathological disorder.

The pathological disorder may be a musculoskeletal disease or disorder,such as muscle atrophy. There are many causes of muscle atrophy,including as a result of treatment with a glucocorticoid such ascortisol, dexamethasone, betamethasone, prednisone, methylprednisolone,or prednisolone. The muscle atrophy can also be a result of denervationdue to nerve trauma or a result of degenerative, metabolic, orinflammatory neuropathy (e.g., Guillian-Barré syndrome, peripheralneuropathy, or exposure to environmental toxins or drugs).

In addition, the muscle atrophy can be a result of myopathy, such asmyotonia; a congenital myopathy, including nemalene myopathy,multi/minicore myopathy and myotubular (centronuclear) myopathy;mitochondrial myopathy; familial periodic paralysis; inflammatorymyopathy; metabolic myopathy, such as caused by a glycogen or lipidstorage disease; dermatomyositisis; polymyositis; inclusion bodymyositis; myositis ossificans; rhabdomyolysis and myoglobinurias.

The myopathy may be caused by a muscular dystrophy syndrome, such asDuchenne, Becker, myotonic, fascioscapulohumeral, Emery-Dreifuss,oculopharyngeal, scapulohumeral, limb girdle, Fukuyama, a congenitalmuscular dystrophy, or hereditary distal myopathy. The musculoskeletaldisease can also be osteoporosis, a bone fracture, short stature, ordwarfism.

In addition, the muscle atrophy can be a result of an adult motor neurondisease, infantile spinal muscular atrophy, amyotrophic lateralsclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathywith multifocal conductor block, paralysis due to stroke or spinal cordinjury, skeletal immobilization due to trauma, prolonged bed rest,voluntary inactivity, involuntary inactivity, metabolic stress ornutritional insufficiency, cancer, AIDS, fasting, a thyroid glanddisorder, diabetes, benign congenital hypotonia, central core disease,burn injury, chronic obstructive pulmonary disease, liver diseases(examples such as fibrosis, cirrhosis), sepsis, renal failure,congestive heart failure, ageing, space travel or time spent in a zerogravity environment.

Examples of age-related conditions that may be treated include,sarcopenia, skin atrophy, muscle wasting, brain atrophy,atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis,osteoarthritis, immunologic incompetence, high blood pressure, dementia,Huntington's disease, Alzheimer's disease, cataracts, age-relatedmacular degeneration, prostate cancer, stroke, diminished lifeexpectancy, frailty, memory loss, wrinkles, impaired kidney function,and age-related hearing loss; metabolic disorders, including Type IIDiabetes, Metabolic Syndrome, hyperglycemia, and obesity.

Other conditions that may be treated with the antibodies of thedisclosure include acute and/or chronic renal disease or failure, liverfibrosis or cirrhosis, cancer such as breast cancer, Parkinson'sDisease; conditions associated with neuronal death, such as ALS, brainatrophy, or dementia and anemia.

Further conditions include cachexia, cachexia associated with arheumatoid arthritis and cachexia associated with cancer.

To date, very few reliable or effective therapies have been developed totreat these disorders.

Based on reported evidence of a role of activins binding to ActRIIBamongst other receptors (Werner and Alzheimer, Cytokine Growth FactorsRev 2006, 17(3):157-171), in contributing to liver, kidney and pulmonaryfibrosis and of a role for myostatin, activins, or ActRIIB in cancers(Tsuchida et al, Endo J, 2008, 55(1):11-21) the antibodies of thedisclosure may be used to treat liver, kidney, pulmonary fibrosis andcancers exemplified by but not restricted to rhabdomyosarcomas,bone-loss inducing cancers, hepatocellular carcinomas, gastrointestinalcancers.

The prevention may be complete, e.g., the total absence of anage-related condition or metabolic disorder. The prevention may also bepartial, such that the likelihood of the occurrence of the age-relatedcondition or metabolic disorder in a subject is less likely to occurthan had the subject not received an antibody of the present disclosure.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” or “signaling activity” refers to abiochemical causal relationship generally initiated by a protein-proteininteraction such as binding of a growth factor to a receptor, resultingin transmission of a signal from one portion of a cell to anotherportion of a cell. In general, the transmission involves specificphosphorylation of one or more tyrosine, serine, or threonine residueson one or more proteins in the series of reactions causing signaltransduction. Penultimate processes typically include nuclear events,resulting in a change in gene expression.

The term ActRIIB or Act IIB receptor refers to human ActRIIB as definedin SEQ ID NO: 181 (AAC64515.1, GI:3769443). Research grade polyclonaland monoclonal anti-ActRIIB antibodies are known in the art, such asthose made by R&D Systems®, MN, USA. Therapeutic anti-ActRIIB antibodieshave not previously been described. Of course, antibodies could beraised against ActRIIB from other species and used to treat pathologicalconditions in those species.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e. “antigen-binding portion”) or singlechains thereof. A naturally occurring “antibody” is a glycoproteincomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as V_(L)) and a lightchain constant region. The light chain constant region is comprised ofone domain, C_(L). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g. effector cells) andthe first component (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g. a portion of ActRIIB). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding region” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g. an isolated antibody that specifically binds ActRIIBis substantially free of antibodies that specifically bind antigensother than ActRIIB). An isolated antibody that specifically bindsActRIIB may, however, have crossreactivity to other antigens, such asActRIIB molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g. human germline sequences, or mutatedversions of human germline sequences or antibody containing consensusframework sequences derived from human framework sequences analysis, forexample, as described in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The human antibodies of the disclosure may include amino acid residuesnot encoded by human sequences (e.g. mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal, e.g.a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g. amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g. from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g. IgM, IgE,IgG such as IgG1 or IgG2) that is provided by the heavy chain constantregion genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, an antibody that “specifically binds to ActRIIBpolypeptide” is intended to refer to an antibody that binds to humanActRIIB polypeptide with a K_(D) of a 100 nM or less, 10 nM or less, 1nM or less. An antibody that “cross-reacts with an antigen other thanActRIIB” is intended to refer to an antibody that binds that antigenwith a K_(D) of 10×10⁻⁹ M or less, 5×10⁻⁹ M or less, or 2×10⁻⁹ M orless. An antibody that “does not cross-react with a particular antigen”is intended to refer to an antibody that binds to that antigen, with aK_(D) of 1.5×10⁻⁸ M or greater, or a K_(D) of 5-10×10⁻⁸ M, or 1×10⁻⁷ Mor greater. In certain embodiments, such antibodies that do notcross-react with the antigen exhibit essentially undetectable bindingagainst these proteins in standard binding assays. K_(D) may bedetermined using a biosensor system, such as a Biacore® system, orSolution Equilibrium Titration.

As used herein, the term “antagonist antibody” is intended to refer toan antibody that inhibits ActRIIB induced signaling activity in thepresence of myostatin. Examples of an assay to detect this includeinhibition of myostatin induced signalling (for instance by a Smaddependent reporter gene assay), inhibition of myostatin induced Smadphosphorylation (P-Smad ELISA) and inhibition of myostatin inducedinhibition of skeletal muscle cell differentiation (for instance by acreatine kinase assay).

In some embodiments, the antibodies inhibit myostatin induced signallingas measured in a Smad dependent reporter gene assay at an IC50 of 10 nMor less, 1 nM or less, or 100 pM or less.

As used herein, an antibody with “no agonistic activity” is intended torefer to an antibody that does not significantly increase ActRIIBmediated signaling activity in the absence of myostatin in a cell-basedassay, such as inhibition of myostatin induced signalling (for instanceby a Smad dependent reporter gene assay), inhibition of myostatininduced Smad phosphorylation (P-Smad ELISA) and inhibition of myostatininduced inhibition of skeletal muscle cell differentiation (for instanceby a creatine kinase assay). Such assays are described in more detailsin the examples below.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d)”, as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, such as the biosensorsystem of Biacore®, or Solution Equilibrium Titration (SET) (see FriguetB et al. (1985) J. Immunol Methods; 77(2): 305-319, and Hanel C et al.(2005) Anal Biochem; 339(1): 182-184).

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalency of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

As used herein, the term “ADCC” or “antibody dependent cellularcytotoxicity” activity refers to human B cell depleting activity. ADCCactivity can be measured by the human B cell depleting assays known inthe art.

In order to get a higher avidity probe, a dimeric conjugate (twomolecules of an antibody protein coupled to a FACS marker) can beconstructed, thus making low affinity interactions (such as with thegermline antibody) more readily detected by FACS. In addition, anothermeans to increase the avidity of antigen binding involves generatingdimers, trimers or multimers of any of the constructs described hereinof the anti-ActRIIB antibodies. Such multimers may be generated throughcovalent binding between individual modules, for example, by imitatingthe natural C-to-N-terminus binding or by imitating antibody dimers thatare held together through their constant regions. The bonds engineeredinto the Fc/Fc interface may be covalent or non-covalent. In addition,dimerizing or multimerizing partners other than Fc can be used inActRIIB hybrids to create such higher order structures. For example, itis possible to use multimerizing domains such as the trimerizing domaindescribed in WO2004/039841 or pentamerizing domain described inWO98/18943.

As used herein, the term “selectivity” for an antibody refers to anantibody that binds to a certain target polypeptide but not to closelyrelated polypeptides.

As used herein, the term “high affinity” for an antibody refers to anantibody having a K_(D) of 1 nM or less for a target antigen. As usedherein, the term “subject” includes any human or nonhuman animal.

The term “nonhuman animal” includes all vertebrates, e.g. mammals andnon-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows,chickens, amphibians, reptiles, etc.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence.

The optimized sequences herein have been engineered to have codons thatare preferred in CHO mammalian cells, however optimized expression ofthese sequences in other eukaryotic cells is also envisioned herein. Theamino acid sequences encoded by optimized nucleotide sequences are alsoreferred to as optimized.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

Standard assays to evaluate the binding ability of the antibodies towardActRIIB of various species are known in the art, including for example,ELISAs, western blots and RIAs. Suitable assays are described in detailin the Examples. The binding affinity of the antibodies also can beassessed by standard assays known in the art, such as by Biacoreanalysis or Solution Equilibrium Titration. Surface plasmon resonancebased techniques such as Biacore can determine the binding kineticswhich allows the calculation of the binding affinity. Assays to evaluatethe effects of the antibodies on functional properties of ActRIIB (e.g.receptor binding, preventing or inducing human B cell proliferation orIgG production) are described in further detail in the Examples.

Accordingly, an antibody that “inhibits” one or more of these ActRIIBfunctional properties (e.g. biochemical, immunochemical, cellular,physiological or other biological activities, or the like) as determinedaccording to methodologies known to the art and described herein, willbe understood to relate to a statistically significant decrease in theparticular activity relative to that seen in the absence of the antibody(e.g. or when a control antibody of irrelevant specificity is present).An antibody that inhibits ActRIIB activity effects such a statisticallysignificant decrease by at least 10% of the measured parameter, by atleast 50%, 80% or 90%, and in certain embodiments an antibody of thedisclosure may inhibit greater than 95%, 98% or 99% of ActRIIBfunctional activity.

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody or otherbinding agent to interfere with the binding of other antibodies orbinding agents to ActRIIB, particularly the ligand binding domain, in astandard competitive binding assay.

The ability or extent to which an antibody or other binding agent isable to interfere with the binding of another antibody or bindingmolecule to ActRIIB, and therefore whether it can be said to cross-blockaccording to the disclosure, can be determined using standardcompetition binding assays. One suitable assay involves the use of theBiacore technology (e.g. by using a BIAcore instrument (Biacore,Uppsala, Sweden)), which can measure the extent of interactions usingsurface plasmon resonance technology. Another assay for measuringcross-blocking uses an ELISA-based approach. A further assay uses FACSanalysis, wherein competition of various antibodies for binding toActRIIB expressing cells is tested (such as described in the Examples).

According to the disclosure, a cross-blocking antibody or other bindingagent according to the disclosure binds to ActRIIB in the describedBIAcore cross-blocking assay such that the recorded binding of thecombination (mixture) of the antibodies or binding agents is between 80%and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding,specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximumtheoretical binding, and more specifically between 70% and 0.1% (e.g.70% to 4%), and more specifically between 65% and 0.1% (e.g. 65% to 4%)of maximum theoretical binding (as defined above) of the two antibodiesor binding agents in combination.

An antibody is defined as cross-blocking an anti-ActRIIB antibody of thedisclosure in an ELISA assay, if the test antibody is able to cause areduction of anti-ActRIIB antibody binding to ActRIIB of between 60% and100%, specifically between 70% and 100%, and more specifically between80% and 100%, when compared to the positive control wells (i.e. the sameanti-ActRIIB antibody and ActRIIB, but no “test” cross-blockingantibody). Examples of cross blocking antibodies as cited herein areMOR08159 and MOR08213. Thus, the disclosure provides antibodies thatcross block MOR08159 or MOR08213 for binding to ActRIIB.

Recombinant Antibodies

Antibodies of the disclosure include the human recombinant antibodies,isolated and structurally characterized, as described in the Examples.The V_(H) amino acid sequences of isolated antibodies of the disclosureare shown in SEQ ID NOs: 99-112. The V_(L) amino acid sequences ofisolated antibodies of the disclosure are shown in SEQ ID NOs: 85-98respectively. Examples of particular full length heavy chain amino acidsequences of antibodies of the disclosure are shown in SEQ ID NOs:146-150 and 156-160. Examples of particular full length light chainamino acid sequences of antibodies of the disclosure are shown in SEQ IDNOs: 141-145 and 151-155 respectively. Other antibodies of thedisclosure include amino acids that have been mutated by amino aciddeletion, insertion or substitution, yet have at least 60, 70, 80, 90,95, 97 or 99 percent sequence identity in the CDR regions with the CDRregions depicted in the sequences described above. In some embodiments,it includes mutant amino acid sequences wherein no more than 1, 2, 3, 4or 5 amino acids have been mutated by amino acid deletion, insertion orsubstitution in the CDR regions when compared with the CDR regionsdepicted in the sequence described above.

Further, variable heavy chain parental nucleotide sequences are shown inSEQ ID NOs: 127-140. Variable light chain parental nucleotide sequencesare shown in SEQ ID NOs: 113-126. Full length light chain nucleotidesequences optimized for expression in a mammalian cell are shown in SEQID NOs: 161-165 and 171-175. Full length heavy chain nucleotidesequences optimized for expression in a mammalian cell are shown in SEQID NOs: 166-170 and 176-180. Other antibodies of the disclosure includeamino acids or nucleic acids that have been mutated, yet have at least60 or more (i.e. 80, 90, 95, 97, 99 or more) percent sequence identityto the sequences described above. In some embodiments, it includesmutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 aminoacids have been mutated by amino acid deletion, insertion orsubstitution in the variable regions when compared with the variableregions depicted in the sequence described above.

Since each of these antibodies binds the same epitope and are progeniesfrom the same parental antibody, the V_(H), V_(L), full length lightchain, and full length heavy chain sequences (nucleotide sequences andamino acid sequences) can be “mixed and matched” to create otheranti-ActRIIB binding molecules of the disclosure. ActRIIB binding ofsuch “mixed and matched” antibodies can be tested using the bindingassays described above and in the Examples (e.g. ELISAs). When thesechains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing should be replaced with a structurally similar V_(H)sequence. Likewise a full length heavy chain sequence from a particularfull length heavy chain/full length light chain pairing should bereplaced with a structurally similar full length heavy chain sequence.Likewise, a V_(L) sequence from a particular V_(H)/V_(L) pairing shouldbe replaced with a structurally similar V_(L) sequence. Likewise a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence. Accordingly, inone aspect, the disclosure provides an isolated recombinant anti-ActRIIBantibody or antigen binding region thereof having: a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 99-112; and a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 85-98.

In another aspect, the disclosure provides:

-   -   (i) an isolated recombinant anti-ActRIIB antibody having: a full        length heavy chain comprising an amino acid sequence selected        from the group consisting of SEQ ID NOs: 99-112; and a full        length light chain comprising an amino acid sequence selected        from the group consisting of SEQ ID NOs: 85-98, or    -   (ii) a functional fragment or functional protein comprising an        antigen binding portion thereof.

In another aspect, the disclosure provides:

-   -   (i) an isolated recombinant anti-ActRIIB antibody having a full        length heavy chain encoded by a nucleotide sequence that has        been optimized for expression in the cell of a mammalian        selected from the group consisting of SEQ ID NOs: 127-140, and a        full length light chain encoded by a nucleotide sequence that        has been optimized for expression in the cell of a mammalian        selected from the group consisting of SEQ ID NOs: 113-126, or    -   (ii) a functional fragment or functional protein comprising an        antigen binding portion thereof.

The amino acid sequences of the V_(H) CDR1s of the antibodies are shownin SEQ ID NOs: 1-14. The amino acid sequences of the V_(H) CDR2s of theantibodies are shown in SEQ ID NOs: 15-28. The amino acid sequences ofthe V_(H) CDR3s of the antibodies are shown in SEQ ID NOs: 29-42. Theamino acid sequences of the V_(L) CDR1s of the antibodies are shown inSEQ ID NOs: 43-56. The amino acid sequences of the V_(L) CDR2s of theantibodies are shown in SEQ ID NOs: 57-70. The amino acid sequences ofthe V_(L) CDR3s of the antibodies are shown in SEQ ID NOs: 71-84. TheCDR regions are delineated using the Kabat system (Kabat, E. A., et al.,1991 Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242). An alternative method of determining CDR regions uses themethod devised by Chothia (Chothia et al. 1989, Nature, 342:877-883).The Chothia definition is based on the location of the structural loopregions. However, due to changes in the numbering system used by Chothia(see e.g. www.biochem.ucl.ac.uk/˜martin/abs/GeneralInfo.html

and www.bioinf org.uk/abs/), this system is now less commonly used.Other systems for defining CDRs exist and are also mentioned in thesetwo websites.

Given that each of these antibodies can bind to ActRIIB and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e. CDRs from differentantibodies can be mixed and matched, each antibody containing a V_(H)CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3 create other anti-ActRIIBbinding molecules of the disclosure. ActRIIB binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g. ELISAs). When V_(H) CDR sequences aremixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particularV_(H) sequence should be replaced with a structurally similar CDRsequence(s). Likewise, when V_(L) CDR sequences are mixed and matched,the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L) sequenceshould be replaced with a structurally similar CDR sequence(s). It willbe readily apparent to the ordinarily skilled artisan that novel V_(H)and V_(L) sequences can be created by substituting one or more V_(H)and/or V_(L) CDR region sequences with structurally similar sequencesfrom the CDR sequences shown herein for monoclonal antibodies of thepresent disclosure.

An isolated recombinant anti-ActRIIB antibody, or antigen binding regionthereof has: a heavy chain variable region CDR1 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-14; a heavychain variable region CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 15-28; a heavy chain variableregion CDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 29-42; a light chain variable region CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 43-56; a light chain variable region CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:57-70; and a light chain variable region CDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 71-84.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 1; a heavy chain variable region CDR2 of SEQ ID NO:15; a heavy chain variable region CDR3 of SEQ ID NO: 29; a light chainvariable region CDR1 of SEQ ID NO: 43; a light chain variable regionCDR2 of SEQ ID NO: 57; and a light chain variable region CDR3 of SEQ IDNO: 71.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 2 a heavy chain variable region CDR2 of SEQ ID NO:16; a heavy chain variable region CDR3 of SEQ ID NO: 30; a light chainvariable region CDR1 of SEQ ID NO: 44; a light chain variable regionCDR2 of SEQ ID NO: 58; and a light chain variable region CDR3 of SEQ IDNO: 72.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQ ID NO:17; a heavy chain variable region CDR3 of SEQ ID NO: 31; a light chainvariable region CDR1 of SEQ ID NO: 45; a light chain variable regionCDR2 of SEQ ID NO: 59; and a light chain variable region CDR3 of SEQ IDNO: 73.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 4; a heavy chain variable region CDR2 of SEQ ID NO:18; a heavy chain variable region CDR3 of SEQ ID NO: 32; a light chainvariable region CDR1 of SEQ ID NO: 46; a light chain variable regionCDR2 of SEQ ID NO: 60; and a light chain variable region CDR3 of SEQ IDNO: 74.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 5; a heavy chain variable region CDR2 of SEQ ID NO:19; a heavy chain variable region CDR3 of SEQ ID NO: 33; a light chainvariable region CDR1 of SEQ ID NO: 47; a light chain variable regionCDR2 of SEQ ID NO: 61; and a light chain variable region CDR3 of SEQ IDNO: 75.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQ ID NO:20; a heavy chain variable region CDR3 of SEQ ID NO: 34; a light chainvariable region CDR1 of SEQ ID NO: 48; a light chain variable regionCDR2 of SEQ ID NO: 62; and a light chain variable region CDR3 of SEQ IDNO: 76.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 7; a heavy chain variable region CDR2 of SEQ ID NO:21; a heavy chain variable region CDR3 of SEQ ID NO: 35; a light chainvariable region CDR1 of SEQ ID NO: 49; a light chain variable regionCDR2 of SEQ ID NO: 63; and a light chain variable region CDR3 of SEQ IDNO: 77.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 8; a heavy chain variable region CDR2 of SEQ ID NO:22; a heavy chain variable region CDR3 of SEQ ID NO: 36; a light chainvariable region CDR1 of SEQ ID NO: 50 a light chain variable region CDR2of SEQ ID NO: 64; and a light chain variable region CDR3 of SEQ ID NO:78.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 9; a heavy chain variable region CDR2 of SEQ ID NO:23; a heavy chain variable region CDR3 of SEQ ID NO: 37; a light chainvariable region CDR1 of SEQ ID NO: 51; a light chain variable regionCDR2 of SEQ ID NO: 65; and a light chain variable region CDR3 of SEQ IDNO: 79.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 10; a heavy chain variable region CDR2 of SEQ ID NO:24; a heavy chain variable region CDR3 of SEQ ID NO: 38; a light chainvariable region CDR1 of SEQ ID NO: 52; a light chain variable regionCDR2 of SEQ ID NO: 66; and a light chain variable region CDR3 of SEQ IDNO: 80.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 11; a heavy chain variable region CDR2 of SEQ ID NO:25; a heavy chain variable region CDR3 of SEQ ID NO: 39; a light chainvariable region CDR1 of SEQ ID NO: 53; a light chain variable regionCDR2 of SEQ ID NO: 67; and a light chain variable region CDR3 of SEQ IDNO: 81.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 12; a heavy chain variable region CDR2 of SEQ ID NO:26; a heavy chain variable region CDR3 of SEQ ID NO: 40; a light chainvariable region CDR1 of SEQ ID NO: 54; a light chain variable regionCDR2 of SEQ ID NO: 68; and a light chain variable region CDR3 of SEQ IDNO: 82.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 13; a heavy chain variable region CDR2 of SEQ ID NO:27; a heavy chain variable region CDR3 of SEQ ID NO: 41; a light chainvariable region CDR1 of SEQ ID NO: 55; a light chain variable regionCDR2 of SEQ ID NO: 69; and a light chain variable region CDR3 of SEQ IDNO: 83.

In one embodiment, the antibody comprises: a heavy chain variable regionCDR1 of SEQ ID NO: 14; a heavy chain variable region CDR2 of SEQ ID NO:28; a heavy chain variable region CDR3 of SEQ ID NO: 42; a light chainvariable region CDR1 of SEQ ID NO: 56; a light chain variable regionCDR2 of SEQ ID NO: 70; and a light chain variable region CDR3 of SEQ IDNO: 84.

In one embodiment, the disclosure provides an antibody comprising: (a)the variable heavy chain sequence of SEQ ID NO: 85 and variable lightchain sequence of SEQ ID NO: 99; (b) the variable heavy chain sequenceof SEQ ID NO: 86 and variable light chain sequence of SEQ ID NO: 100;(c) the variable heavy chain sequence of SEQ ID NO: 87 and variablelight chain sequence of SEQ ID NO: 101; (d) the variable heavy chainsequence of SEQ ID NO: 88 and variable light chain sequence of SEQ IDNO: 102; (e) the variable heavy chain sequence of SEQ ID NO: 89 andvariable light chain sequence of SEQ ID NO: 103; (0 the variable heavychain sequence of SEQ ID NO: 90 and variable light chain sequence of SEQID NO: 104; (g) the variable heavy chain sequence of SEQ ID NO: 91 andvariable light chain sequence of SEQ ID NO: 105; (h) the variable heavychain sequence of SEQ ID NO: 92 and variable light chain sequence of SEQID NO: 106; (i) the variable heavy chain sequence of SEQ ID NO: 93 andvariable light chain sequence of SEQ ID NO: 107; (j) the variable heavychain sequence of SEQ ID NO: 94 and variable light chain sequence of SEQID NO: 108; (k) the variable heavy chain sequence of SEQ ID NO: 95 andvariable light chain sequence of SEQ ID NO: 109; (1) the variable heavychain sequence of SEQ ID NO: 96 and variable light chain sequence of SEQID NO: 110; (m) the variable heavy chain sequence of SEQ ID NO: 97 andvariable light chain sequence of SEQ ID NO: 111; or (n) the variableheavy chain sequence of SEQ ID NO: 98 and variable light chain sequenceof SEQ ID NO: 112.

In one embodiment, the disclosure provides an antibody comprising: (a)the heavy chain sequence of SEQ ID NO: 146 and light chain sequence ofSEQ ID NO: 141; (b) the heavy chain sequence of SEQ ID NO: 147 and lightchain sequence of SEQ ID NO: 142; (c) the heavy chain sequence of SEQ IDNO: 148 and light chain sequence of SEQ ID NO: 143; (d) the heavy chainsequence of SEQ ID NO: 149 and light chain sequence of SEQ ID NO: 144;(e) the heavy chain sequence of SEQ ID NO: 150 and light chain sequenceof SEQ ID NO: 145; (f) the heavy chain sequence of SEQ ID NO: 156 andlight chain sequence of SEQ ID NO: 151; (g) the heavy chain sequence ofSEQ ID NO: 157 and light chain sequence of SEQ ID NO: 152; (h) the heavychain sequence of SEQ ID NO: 158 and light chain sequence of SEQ ID NO:153; (i) the heavy chain sequence of SEQ ID NO: 159 and light chainsequence of SEQ ID NO: 154; or (j) the heavy chain sequence of SEQ IDNO: 160 and light chain sequence of SEQ ID NO: 155.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e. greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g. murine germline sequences). In certaincases, a human antibody may be at least 80%, 90%, or at least 95%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence tothe amino acid sequence encoded by the germline immunoglobulin gene.Typically, a human antibody derived from a particular human germlinesequence will display no more than 10 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene.In certain cases, the human antibody may display no more than 5, or evenno more than 4, 3, 2, or 1 amino acid difference from the amino acidsequence encoded by the germline immunoglobulin gene.

In one embodiment the antibody of the disclosure is that encoded bypBW522 or pBW524 (deposited at DSMZ, Inhoffenstr. 7B, D-38124Braunschweig, Germany on 18 Aug. 2009 under deposit numbers DSM22873 andDSM22874, respectively).

Homologous Antibodies

In yet another embodiment, an antibody of the disclosure has full lengthheavy and light chain amino acid sequences; full length heavy and lightchain nucleotide sequences, variable region heavy and light chainnucleotide sequences, or variable region heavy and light chain aminoacid sequences that are homologous to the amino acid and nucleotidesequences of the antibodies described herein, and wherein the antibodiesretain the desired functional properties of the anti-ActRIIB antibodiesof the disclosure.

For example, the disclosure provides an isolated recombinantanti-ActRIIB antibody (or a functional fragment or functional proteincomprising an antigen binding portion thereof) comprising a heavy chainvariable region and a light chain variable region, wherein: the heavychain variable region comprises an amino acid sequence that is at least80%, or at least 90% (in various embodiments, at least 95, 97 or 99%)identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 99-112; the light chain variable region comprises anamino acid sequence that is at least 80%, or at least 90% (in variousembodiments, at least 95, 97 or 99%) identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 85-98; and theantibody exhibits at least one of the following functional properties:(i) it inhibits myostatin binding in vitro or in vivo and/or (ii)decreases inhibition of muscle differentiation through theSmad-dependent pathway.

In a further example, the disclosure provides an isolated recombinantanti-ActRIIB antibody, (or a functional fragment or functional proteincomprising an antigen binding portion thereof) comprising a full lengthheavy chain and a full length light chain, wherein: the full lengthheavy chain comprises an amino acid sequence that is at least 80%, or atleast 90% (in various embodiments, at least 95, 97 or 99%) identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:146-150 and 156-160; the full length light chain comprises an amino acidsequence that is at least 80%, or at least 90% (in various embodiments,at least 95, 97 or 99%) identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 141-145 and 151-155; and theantibody exhibits at least one of the following functional properties:(i) it inhibits myostatin binding in vitro or in vivo and/or (ii)decreases inhibition of muscle differentiation through theSmad-dependent pathway. In one embodiment, such an antibody binds to theligand binding domain of ActRIIB.

In another example, the disclosure provides an isolated recombinantanti-ActRIIB antibody (or a functional fragment or functional proteincomprising an antigen binding portion thereof), comprising a full lengthheavy chain and a full length light chain, wherein: the full lengthheavy chain is encoded by a nucleotide sequence that is at least 80%, orat least 90% (in various embodiments, at least 95, 97 or 99%) identicalto a nucleotide sequence selected from the group consisting of SEQ IDNOs: 166-170 and 176-180; the full length light chain is encoded by anucleotide sequence that is at least 80%, or at least 90% (in variousembodiments, at least 95, 97 or 99%) identical to a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 161-165 and 171-175;and the antibody exhibits at least one of the following functionalproperties: (i) it inhibits myostatin binding in vitro or in vivo and/or(ii) decreases inhibition of muscle differentiation through theSmad-dependent pathway. In various embodiments such an antibody binds tothe ligand binding domain of ActRIIB.

In various embodiments, the antibody may exhibit one or more, two ormore, or three of the functional properties discussed above. Theantibody can be, for example, a human antibody, a humanized antibody ora chimeric antibody. In various embodiments, the antibody is a fullyhuman IgG1 antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forthabove. In other embodiments, the V_(H) and/or V_(L) amino acid sequencesmay be identical except an amino acid substitution in no more than 1, 2,3, 4 or 5 amino acid position. An antibody having V_(H) and V_(L)regions having high (i.e. 80% or greater) sequence identity to the V_(H)and V_(L) regions of SEQ ID NOs 99-112 and SEQ ID NOs: 85-98respectively, can be obtained by mutagenesis (e.g. site-directed orPCR-mediated mutagenesis) of nucleic acid molecules SEQ ID NOs: 127-140and 113-126 respectively, followed by testing of the encoded alteredantibody for retained function (i.e. the functions set forth above)using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain amino acid sequences may be 80%, 90%, 95%, 96%, 97%, 98% or99% identical to the sequences set forth above. An antibody having afull length heavy chain and full length light chain having high (i.e.80% or greater) identity to the full length heavy chains of any of SEQID NOs: 146-150 and 156-160 and full length light chains of any of SEQID NOs: 141-145 and 151-155 respectively, can be obtained by mutagenesis(e.g. site-directed or PCR-mediated mutagenesis) of nucleic acidmolecules SEQ ID NOs: 166-170 and 176-180 and SEQ ID NOs: 161-165 and171-175 respectively, followed by testing of the encoded alteredantibody for retained function (i.e. the functions set forth above)using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain nucleotide sequences may be 80%, 90%, 95%, 96%, 97%, 98% or99% identical to the sequences set forth above.

In other embodiments, the variable regions of heavy chain and/or lightchain nucleotide sequences may be 80%, 90%, 95%, 96%, 97%, 98% or 99%identical to the sequences set forth above.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e. % identity=# of identical positions/total # of positions ×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the disclosure has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-ActRIIB antibodies of the disclosure.Accordingly, the disclosure provides an isolated recombinantanti-ActRIIB antibody, or a functional fragment or functional proteincomprising an antigen binding portion thereof, consisting of a heavychain variable region comprising CDR1, CDR2, and CDR3 sequences and alight chain variable region comprising CDR1, CDR2, and CDR3 sequences,wherein: the heavy chain variable region CDR1 amino acid sequences areselected from the group consisting of SEQ ID NOs: 1-14, and conservativemodifications thereof, the heavy chain variable region CDR2 amino acidsequences are selected from the group consisting of SEQ ID NOs: 15-28,and conservative modifications thereof, the heavy chain variable regionCDR3 amino acid sequences are selected from the group consisting of SEQID NOs: 29-42, and conservative modifications thereof; the light chainvariable regions CDR1 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 43-56, and conservative modifications thereof,the light chain variable regions CDR2 amino acid sequences are selectedfrom the group consisting of SEQ ID NOs: 57-70, and conservativemodifications thereof; the light chain variable regions of CDR3 aminoacid sequences are selected from the group consisting of SEQ ID NOs:71-84, and conservative modifications thereof. In various embodiments,the antibody exhibits at least one of the following functionalproperties: (i) it inhibits myostatin binding in vitro or in vivo and/or(ii) decreases inhibition of muscle differentiation through theSmad-dependent pathway.

In various embodiments, the antibody may exhibit one or both of thefunctional properties listed above. Such antibodies can be, for example,human antibodies, humanized antibodies or chimeric antibodies.

In other embodiments, an antibody of the disclosure optimized forexpression in a mammalian cell has a full length heavy chain sequenceand a full length light chain sequence, wherein one or more of thesesequences have specified amino acid sequences based on the antibodiesdescribed herein or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the anti-ActRIIBantibodies of the disclosure. Accordingly, the disclosure provides anisolated monoclonal anti-ActRIIB antibody optimized for expression in amammalian cell consisting of a full length heavy chain and a full lengthlight chain wherein: the full length heavy chain has amino acidsequences selected from the group of SEQ ID NOs: 146-150 and 156-160,and conservative modifications thereof, and the full length light chainhas amino acid sequences selected from the group of SEQ ID NOs: 141-145and 151-155, and conservative modifications thereof; and the antibodyexhibits at least one of the following functional properties: (i) itinhibits myostatin binding in vitro or in vivo and/or (ii) decreasesinhibition of muscle differentiation through the Smad-dependent pathway.

In various embodiments, the antibody may exhibit one or both of thefunctional properties listed above. Such antibodies can be, for example,human antibodies, humanized antibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the disclosure by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g. lysine, arginine, histidine), acidic side chains (e.g.aspartic acid, glutamic acid), uncharged polar side chains (e.g.glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g. alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g. threonine, valine, isoleucine) and aromatic side chains(e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, one or moreamino acid residues within the CDR regions of an antibody of thedisclosure can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-ActRIIB Antibodies

In another embodiment, the disclosure provides antibodies that bind tothe same epitope as the various specific anti-ActRIIB antibodies of thedisclosure described herein. All the antibodies described in theExamples that are capable of blocking myostatin binding to ActRIIB bindthe same epitope in ActRIIB with high affinity, said epitope beingcomprised between amino acids 19-134 of SEQ ID NO:181.

Additional antibodies can therefore be identified based on their abilityto cross-compete (e.g. to competitively inhibit the binding of, in astatistically significant manner) with other antibodies of thedisclosure in standard ActRIIB binding assays. The ability of a testantibody to inhibit the binding of antibodies of the present disclosureto human ActRIIB demonstrates that the test antibody can compete withthat antibody for binding to human ActRIIB; such an antibody may,according to non-limiting theory, bind to the same or a related (e.g. astructurally similar or spatially proximal) epitope on human ActRIIB asthe antibody with which it competes. In a certain embodiment, theantibody that binds to the same epitope on human ActRIIB as theantibodies of the present disclosure is a human recombinant antibody.Such human recombinant antibodies can be prepared and isolated asdescribed in the Examples.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 85, and the variable light chain sequence recitedin SEQ ID NO: 99.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 86, and the variable light chain sequence recitedin SEQ ID NO: 100.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 87, and the variable light chain sequence recitedin SEQ ID NO: 101.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 88, and the variable light chain sequence recitedin SEQ ID NO: 102.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 89, and the variable light chain sequence recitedin SEQ ID NO: 103.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 90, and the variable light chain sequence recitedin SEQ ID NO: 104.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 91, and the variable light chain sequence recitedin SEQ ID NO: 105.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 92, and the variable light chain sequence recitedin SEQ ID NO: 106.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 93, and the variable light chain sequence recitedin SEQ ID NO: 107.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 94, and the variable light chain sequence recitedin SEQ ID NO: 108.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 95, and the variable light chain sequence recitedin SEQ ID NO: 109.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 96, and the variable light chain sequence recitedin SEQ ID NO: 110.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 97, and the variable light chain sequence recitedin SEQ ID NO: 111.

Thus, the disclosure provides an antibody that binds to an epitoperecognised by an antibody having the variable heavy chain sequencerecited in SEQ ID NO: 98, and the variable light chain sequence recitedin SEQ ID NO: 112.

Following more detailed epitope mapping experiments, the binding regionsof particular antibodies of the disclosure have been more clearlydefined.

Thus, the disclosure provides an antibody that binds to an epitopecomprising amino acids 78-83 of SEQ ID NO: 181 (WLDDFN—SEQ ID NO:188).

The disclosure also provides an antibody that binds to an epitopecomprising amino acids 76-84 of SEQ ID NO: 181 (GCWLDDFNC—SEQ IDNO:186).

The disclosure also provides an antibody that binds to an epitopecomprising amino acids 75-85 of SEQ ID NO: 181 (KGCWLDDFNCY—SEQ IDNO:190).

The disclosure also provides an antibody that binds to an epitopecomprising amino acids 52-56 of SEQ ID NO: 181 (EQDKR—SEQ ID NO:189).

The disclosure also provides an antibody that binds to an epitopecomprising amino acids 49-63 of SEQ ID NO: 181 (CEGEQDKRLHCYASW—SEQ IDNO:187).

The disclosure also provides antibodies that bind to epitopes consistingof these sequences or epitopes comprising combinations of these epitoperegions.

Thus, the disclosure also provides an antibody that binds to an epitopecomprising or consisting of amino acids 78-83 of SEQ ID NO: 181 (WLDDFN)and amino acids 52-56 of SEQ ID NO: 181 (EQDKR).

Engineered and Modified Antibodies

An antibody of the disclosure further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e. V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g. Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.).

Accordingly, another embodiment of the disclosure pertains to anisolated monoclonal anti-ActRIIB antibody, or a functional fragment orfunctional protein comprising an antigen binding portion thereof,comprising a heavy chain variable region comprising CDR1 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 1-14; CDR2 sequences having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 15-28; CDR3 sequences having anamino acid sequence selected from the group consisting of SEQ ID NOs:29-42, respectively; and a light chain variable region having CDR1sequences having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 43-56; CDR2 sequences having an amino acidsequence selected from the group consisting of SEQ ID NOs: 57-70; andCDR3 sequences consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 71-84, respectively. Thus, suchantibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies, yet may contain different framework sequences from theseantibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al., [supra]; Tomlinson, I. M., et al., 1992J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836.

An example of framework sequences for use in the antibodies of thedisclosure are those that are structurally similar to the frameworksequences used by selected antibodies of the disclosure, e.g. consensussequences and/or framework sequences used by monoclonal antibodies ofthe disclosure. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2and 3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g. affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the disclosure provides isolatedanti-ActRIIB monoclonal antibodies, or a functional fragment orfunctional protein comprising an antigen binding portion thereof,consisting of a heavy chain variable region having: a V_(H) CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 1-14 or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 1-14; a V_(H) CDR2 region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 15-28, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 15-28; a V_(H) CDR3region having an amino acid sequence selected from the group consistingof SEQ ID NOs: 29-42, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 29-42; a V_(L) CDR1 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 43-56, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 43-56;a V_(L) CDR2 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 52-70, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 52-70; and a V_(L) CDR3 regionhaving an amino acid sequence selected from the group consisting of SEQID NOs: 71-84, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 71-84.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to ActRIIB. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof (such as those disclosed elsewhere herein), andinclude immunoglobulins of other animal species, for example, havinghumanized aspects. Single heavy-chain antibodies such as thoseidentified in camelids are of particular interest in this regard. Novelframeworks, scaffolds and fragments continue to be discovered anddeveloped by those skilled in the art.

In one aspect, the disclosure pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe disclosure can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target protein of SEQ ID NO: 181 (invarious embodiments, the ligand binding domain thereof as shown in SEQID NO: 182). Such compounds are known herein as “polypeptides comprisinga target-specific binding region”. Examples of non-immunoglobulinframework are further described in the sections below (camelidantibodies and non-antibody scaffold).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedaryfamily (Camelus bactrianus and Camelus dromaderius) including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from other animals(see WO94/04678).

A region of the camelid antibody which is the small single variabledomain identified as V_(HH) can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody” (see U.S. Pat. No. 5,759,808; Stijlemans, B. et al., 2004 JBiol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788;Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448;Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys,M. et al. 1998 EMBO J 17: 3512-3520). Engineered libraries of camelidantibodies and antibody fragments are commercially available, forexample, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e. the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e. camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitate drugtransport across the blood brain barrier (see US2004/0161738). Thesefeatures combined with the low antigenicity to humans indicate greattherapeutic potential. Further, these molecules can be fully expressedin prokaryotic cells such as E. coli and are expressed as fusionproteins with bacteriophage and are functional.

Accordingly, a feature of the present disclosure is a camelid antibodyor nanobody having high affinity for ActRIIB. In certain embodimentsherein, the camelid antibody or nanobody is naturally produced in thecamelid animal, i.e. is produced by the camelid following immunizationwith ActRIIB or a peptide fragment thereof, using techniques describedherein for other antibodies. Alternatively, the anti-ActRIIB camelidnanobody is engineered, i.e. produced by selection for example from alibrary of phage displaying appropriately mutagenized camelid nanobodyproteins using panning procedures with ActRIIB as a target as describedin the examples herein. Engineered nanobodies can further be customizedby genetic engineering to have a half life in a recipient subject offrom 45 minutes to two weeks.

In a specific embodiment, the camelid antibody or nanobody is obtainedby grafting the CDRs sequences of the heavy or light chain of the humanantibodies of the disclosure into nanobody or single domain antibodyframework sequences, as described for example in WO94/04678.

Non-Antibody Scaffold

Known non-immunoglobulin frameworks or scaffolds include, but are notlimited to, Adnectins (fibronectin) (Compound Therapeutics, Inc.,Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland),domain antibodies (Domantis, Ltd (Cambridge, Mass.) and Ablynx nv(Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.(Mountain View, Calif.)), Protein A (Affibody AG, Sweden) and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany),protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

(i) Fibronectin Scaffold

The fibronectin scaffolds are based in various embodiments onfibronectin type III domain (e.g. the tenth module of the fibronectintype III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8beta strands which are distributed between two beta sheets, whichthemselves pack against each other to form the core of the protein, andfurther containing loops (analogous to CDRs) which connect the betastrands to each other and are solvent exposed. There are at least threesuch loops at each edge of the beta sheet sandwich, where the edge isthe boundary of the protein perpendicular to the direction of the betastrands (U.S. Pat. No. 6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the disclosure using standardcloning techniques.

(ii) Ankyrin—Molecular Partners

The technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel α-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay.

(iii) Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example,US2004/0175756; US2005/0053973; US2005/0048512; and US2006/0008844.

(vi) Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g. U.S. Pat. No. 5,831,012). Affibody®molecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® molecules issimilar to that of an antibody.

(v) Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target molecules of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is WO1999/16873.

(vi) Affilin—Scil Proteins

Affilin™ molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New Affilin™ molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulinproteins. Scil Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO2001/004144 and examples of“ubiquitin-like” proteins are described in WO2004/106368.

(vii) Protein Epitope Mimetics (PEM)

PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2 kDa)mimicking beta-hairpin secondary structures of proteins, the majorsecondary structure involved in protein-protein interactions.

Framework or Fc Engineering

Engineered antibodies of the disclosure include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed by the disclosure.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in US2003/0153043.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the disclosure may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the disclosure may bechemically modified (e.g. one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, toincrease the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. No. 5,869,046 and U.S. Pat. No. 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, both by Winter etal. In particular, residues 234 and 235 may be mutated. In particular,these mutations may be to alanine. Thus in one embodiment the antibodyof the disclosure has a mutation in the Fc region at one or both ofamino acids 234 and 235. In another embodiment, one or both of aminoacids 234 and 235 may be substituted to alanine. Substitution of bothamino acids 234 and 235 to alanine results in a reduced ADCC activity.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered Clq binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in WO94/29351.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in WO00/42072. Moreover, the binding sites on humanIgG1 for FcγRl, FcγRII, FcγRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields, R. L. et al.,2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e. theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for the antigen. Suchcarbohydrate modifications can be accomplished by; for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the disclosure to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. Therefore, in one embodiment, theantibodies of the disclosure are produced by recombinant expression in acell line which exhibit hypofucosylation pattern, for example, amammalian cell line with deficient expression of the FUT8 gene encodingfucosyltransferase. WO03/035835 describes a variant CHO cell line, Lec13cells, with reduced ability to attach fucose to Asn(297)-linkedcarbohydrates, also resulting in hypofucosylation of antibodiesexpressed in that host cell (see also Shields, R. L. et al., 2002 J.Biol. Chem. 277:26733-26740). WO99/54342 describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).Alternatively, the antibodies of the disclosure can be produced in ayeast or a filamentous fungi engineered for mammalian-like glycosylationpattern, and capable of producing antibodies lacking fucose asglycosylation pattern (see for example EP 1297172B1).

Another modification of the antibodies herein that is contemplated bythe disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g. serum) half-life of the antibody.To pegylate an antibody, the antibody, or fragment thereof, typically isreacted with polyethylene glycol (PEG), such as a reactive ester oraldehyde derivative of PEG, under conditions in which one or more PEGgroups become attached to the antibody or antibody fragment. Thepegylation can be carried out by an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). As used herein, the term “polyethylene glycol”is intended to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the disclosure (see for example, EP0154316 andEP0401384).

Another modification of the antibodies that is contemplated by thedisclosure is a conjugate or a protein fusion of at least theantigen-binding region of the antibody of the disclosure to serumprotein, such as human serum albumin or a fragment thereof to increasehalf-life of the resulting molecule (see, for example, EP0322094).

Another possibility is a fusion of at least the antigen-binding regionof the antibody of the disclosure to proteins capable of binding toserum proteins, such human serum albumin to increase half life of theresulting molecule (see, for example, EP0486525).

Methods of Engineering Altered Antibodies

As discussed above, the anti-ActRIIB antibodies having V_(H) and V_(L)sequences or full length heavy and light chain sequences shown hereincan be used to create new anti-ActRIIB antibodies by modifying fulllength heavy chain and/or light chain sequences, V_(H) and/or V_(L)sequences, or the constant region(s) attached thereto. Thus, in anotheraspect of the disclosure, the structural features of an anti-ActRIIBantibody of the disclosure are used to create structurally relatedanti-ActRIIB antibodies that retain at least one functional property ofthe antibodies of the disclosure, such as binding to human ActRIIB butalso inhibit one or more functional properties of ActRIIB (for example,the inhibition of Smad activation).

For example, one or more CDR regions of the antibodies of the presentdisclosure, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-ActRIIB antibodies of the disclosure, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e. express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the disclosure provides a method forpreparing an anti-ActRIIB antibody consisting of: a heavy chain variableregion antibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 1-14, a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 15-28 and/or a CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 29-42; and a light chain variable regionantibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 43-56, a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 57-70 and/or a CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 71-84; altering at least one amino acidresidue within the heavy chain variable region antibody sequence and/orthe light chain variable region antibody sequence to create at least onealtered antibody sequence; and expressing the altered antibody sequenceas a protein.

Accordingly, in another embodiment, the disclosure provides a method forpreparing an anti-ActRIIB antibody optimized for expression in amammalian cell consisting of: a full length heavy chain antibodysequence having a sequence selected from the group of SEQ ID NOs:146-150 and 156-160; and a full length light chain antibody sequencehaving a sequence selected from the group of SEQ ID NOs: 141-145 and151-155; altering at least one amino acid residue within the full lengthheavy chain antibody sequence and/or the full length light chainantibody sequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences selected among the groupconsisting of SEQ ID NO: 29-42 and SEQ ID NO: 71-84 or minimal essentialbinding determinants as described in US2005/0255552 and diversity onCDR1 and CDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the anti-ActRIIB antibodies described herein,which functional properties include, but are not limited to,specifically binding to human ActRIIB and inhibition of Smad activation.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g. ELISAs).

In certain embodiments of the methods of engineering antibodies of thedisclosure, mutations can be introduced randomly or selectively alongall or part of an anti-ActRIIB antibody coding sequence and theresulting modified anti-ActRIIB antibodies can be screened for bindingactivity and/or other functional properties as described herein.Mutational methods have been described in the art. For example,WO02/092780 describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, WO03/074679 describes methods ofusing computational screening methods to optimize physiochemicalproperties of antibodies.

Nucleic Acid Molecules Encoding Particular Antibodies

Another aspect of the disclosure pertains to nucleic acid molecules thatencode the antibodies of the disclosure. Examples of full length lightchain nucleotide sequences optimized for expression in a mammalian cellare shown in SEQ ID NOs: 161-165 and 171-175. Examples of full lengthheavy chain nucleotide sequences optimized for expression in a mammaliancell are shown in SEQ ID NOs: 166-170 and 176-180.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants, e.g.other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of thedisclosure can be, for example, DNA or RNA and may or may not containintronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector. The disclosure alsoprovides the vectors referred to as pBW522 and pBW524 (deposited atDSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 18 Aug. 2009under deposit numbers DSM22873 and DSM22874, respectively).

Nucleic acids of the disclosure can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g. using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromvarious phage clones that are members of the library.

Also included within the scope of the disclosure are variant nucleicacid sequences that comprise one or more deletions, additions orsubstitutions. In one embodiment, the disclosure comprises one or moreof SEQ ID NOs: 113-140 or 161-180, which comprises a conservativenucleotide substitution. Due to the degeneracy of the genetic code, anamino acid may be encoded by more than one codon. Thus, it is possibleto amend the nucleotide sequence, while the translated amino acidsequence remains the same.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to an scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA molecule, or to a fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked”, as used in this context, isintended to mean that the two DNA fragments are joined in a functionalmanner, for example, such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame, or such that the protein is expressedunder control of a desired promoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g. Kabat, E. A., et al. [supra]) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments,the heavy chain constant region is selected among IgG1 isotypes. For aFab fragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g. Kabat,E. A., et al. [supra]) and DNA fragments encompassing these regions canbe obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (see e.g.Bird et al., 1988 Science 242:423-426; Huston et al., 1988 Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature348:552-554).

The nucleic acids of the disclosure may be used in gene delivery. Thatis, nucleic acid encoding the polypeptides (antibodies or functionalproteins) of the disclosure may be directly delivered to a patient fortranslation in the patient.

The nucleic acid is typically “packaged” for administration to apatient. Gene delivery vehicles may be non-viral, such as liposomes, orreplication-deficient viruses, such as adenovirus as described byBerkner, K. L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) oradeno-associated virus (AAV) vectors as described by Muzyczka, N., inCurr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Pat. No.5,252,479. Alternatively a retrovirus, such as a lentivirus may be used.For example, a nucleic acid molecule encoding a polypeptide of thedisclosure may be engineered for expression in a replication-defectiveretroviral vector. This expression construct may then be isolated andintroduced into a packaging cell transduced with a retroviral plasmidvector containing RNA encoding the polypeptide, such that the packagingcell now produces infectious viral particles containing the gene ofinterest. These producer cells may be administered to a subject forengineering cells in vivo and expression of the polypeptide in vivo (seeChapter 20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics(1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).

Another approach is the administration of “naked DNA” in which thetherapeutic gene is directly injected into the bloodstream or muscletissue.

Generation of Particular Monoclonal Antibodies

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g. the standardsomatic cell hybridization technique of Kohler and Milstein (1975 Nature256: 495). Many techniques for producing monoclonal antibody can beemployed e.g. viral or oncogenic transformation of B lymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present disclosure can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g. human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g. U.S.Pat. No. 4,816,567). To create a humanized antibody, the murine CDRregions can be inserted into a human framework using methods known inthe art (see e.g. U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089;5,693,762 and 6,180,370).

In a certain embodiment, the antibodies of the disclosure are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstActRIIB can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb mouse (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g. Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 [supra]; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et al., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and5,545,807; as well as WO92/103918, WO93/12227, WO94/25585, WO97/113852,WO98/24884; WO99/45962; and WO01/14424.

In another embodiment, human antibodies of the disclosure can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in WO02/43478.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-ActRIIB antibodies of the disclosure. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g. U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-ActRIIB antibodies of the disclosure. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise anti-ActRIIBantibodies of the disclosure.

Human recombinant antibodies of the disclosure can also be preparedusing phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art or described in the examplesbelow. See for example: U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,698;5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081. Human monoclonalantibodies of the disclosure can also be prepared using SCID mice intowhich human immune cells have been reconstituted such that a humanantibody response can be generated upon immunization. Such mice aredescribed in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767.

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies of thedisclosure, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×145in flat bottom microtiter plates, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0:055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia). Eluted IgG can bechecked by gel electrophoresis and high performance liquidchromatography to ensure purity. The buffer solution can be exchangedinto PBS, and the concentration can be determined by OD₂₈₀ using 1.43extinction coefficient. The monoclonal antibodies can be aliquoted andstored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the disclosure also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g. Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g. PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g. ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the CH segment(s) within the vector andthe V_(L) segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e. asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g. polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g. theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g. origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g. U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Selectable marker genes include the dihydrofolatereductase (DHFR) gene (for use in dhfr− host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g. electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the disclosure in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, inparticular mammalian host cells, is discussed because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today6:12-13).

Mammalian host cells for expressing the recombinant antibodies of thedisclosure include Chinese Hamster Ovary (CHO cells) (including dhfr−CHO cells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g. as described inR. J. Kaufman and P. A. Sharp, 1982 Mol. Biol. 159:601-621), NSO myelomacells, COS cells and SP2 cells. In one embodiment the host cells are CHOK1PD cells. In particular, for use with NSO myeloma cells, anotherexpression system is the GS gene expression system shown in WO87/04462,WO89/01036 and EP 338,841. In one embodiment, mammalian host cells forexpressing the recombinant antibodies of the disclosure includemammalian cell lines deficient for FUT8 gene expression, for example asdescribed in U.S. Pat. No. 6,946,292B2. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods.

Immunoconjugates

In another aspect, the present disclosure features an anti-ActRIIBantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g. an immunosuppressant) or a radiotoxin.Such conjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g. kills) cells.

Cytotoxins can be conjugated to antibodies of the disclosure usinglinker technology available in the art. Examples of linker types thathave been used to conjugate a cytotoxin to an antibody include, but arenot limited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases, in variousembodiments, expressed in tumor tissue such as cathepsins (e.g.cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.,2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al., 2003 CancerImmunol. Immunother. 52:328-337; Payne, G. 2003 Cancer Cell 3:207-212;Allen, T. M., 2002 Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman,R. J., 2002 Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P. D. andSpringer, C. J., 2001 Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present disclosure also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Methods for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (DEC Pharmaceuticals) andBexxar (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the disclosure.

The antibody conjugates of the disclosure can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g. Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Inmunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present disclosure features bispecific ormultispecific molecules comprising an anti-ActRIIB antibody, or afragment thereof, of the disclosure. An antibody of the disclosure, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g. another peptide or protein (e.g. anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the disclosure may in fact be derivatized or linked tomore than one other functional molecule to generate multi-specificmolecules that bind to more than two different binding sites and/ortarget molecules; such multi-specific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the disclosure, an antibody of the disclosurecan be functionally linked (e.g. by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for ActRIIB and asecond binding specificity for a second target epitope. For example, thesecond target epitope may be another epitope of ActRIIB different fromthe first target epitope.

Additionally, for the disclosure in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the disclosure compriseas a binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g. an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

Other antibodies which can be employed in the bispecific molecules ofthe disclosure are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g. Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule of thedisclosure can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g. growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g. an antibody) specific forthe complex of interest.

Multivalent Antibodies

In another aspect, the present disclosure provides multivalentantibodies comprising at least two identical or differentantigen-binding portions of the antibodies of the disclosure binding toActRIIB. In one embodiment, the multivalent antibodies provides at leasttwo, three or four antigen-binding portions of the antibodies. Theantigen-binding portions can be linked together via protein fusion orcovalent or non covalent linkage. Alternatively, methods of linkage havebeen described for the bispecific molecules. Tetravalent compounds canbe obtained for example by cross-linking antibodies of the antibodies ofthe disclosure with an antibody that binds to the constant regions ofthe antibodies of the disclosure, for example the Fc or hinge region.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g. apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent disclosure, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g. two or more different) antibodies, or immunoconjugates orbispecific molecules of the disclosure. For example, a pharmaceuticalcomposition of the disclosure can comprise a combination of antibodiesthat bind to different epitopes on the target antigen or that havecomplementary activities.

Pharmaceutical compositions of the disclosure also can be administeredin combination therapy, i.e. combined with other agents. For example,the combination therapy can include an anti-ActRIIB antibody of thepresent disclosure combined with at least one other muscle mass/strengthincreasing agent, for example, IGF-1, IGF-2 or variants of IGF-1 orIGF-2, an anti-myostatin antibody, a myostatin propeptide, a myostatindecoy protein that binds ActRIIB but does not activate it, a beta 2agonist, a Ghrelin agonist, a SARM, GH agonists/mimetics or follistatin.Examples of therapeutic agents that can be used in combination therapyare described in greater detail below in the section on uses of theantibodies of the disclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Depending onthe route of administration, the active compound, i.e. antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the disclosure may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g. Berge, S. M., et al., 1977 J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, one can include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption for example, monostearatesalts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of agents enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other agents from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive agent plus any additional desired agent from a previouslysterile-filtered solution thereof.

The amount of active agent which can be combined with a carrier materialto produce a single dosage form will vary depending upon the subjectbeing treated, and the particular mode of administration. The amount ofactive agent which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.01 percent to aboutninety-nine percent of active agent, from about 0.1 percent to about 70percent, or from about 1 percent to about 30 percent of active agent incombination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g. a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe ranges of 1-10 mg/kg or 3-7 mg/kg. An example treatment regimeentails administration once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months. Alternatively, the antibody may beadministered about once a year or once only. Such administration may becarried out intraveneously or subcutaneously. Dosage regimens for ananti-ActRIIB antibody of the disclosure include 1 mg/kg body weight or 3mg/kg body weight by intravenous administration, with the antibody beinggiven using one of the following dosing schedules: every four weeks forsix dosages, then every three months; every three weeks; 3 mg/kg bodyweight once followed by 1 mg/kg body weight every three weeks.

The dosage should be one that causes an upregulation of muscle massand/or strength. In various embodiments the effect is on skeletalmuscle. In various embodiments, the dosage causes muscle hypertrophywith no more than a proportional increase in the size of internal organs(e.g. heart, lungs, liver, kidneys). Such a proportional increase may becompared by measuring either mass or volume.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths, every six months or yearly. Intervals can also be irregular asindicated by measuring blood levels of antibody to the target antigen inthe patient. In some methods, dosage is adjusted to achieve a plasmaantibody concentration of about 1-1000 μg/ml and in some methods about25-300 μg/ml. For example, an ActRIIB antibody of the disclosure couldbe co-administered with an anti-myostatin antibody.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active agents in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active agent which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present disclosureemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-ActRIIB antibody of thedisclosure can result in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease afflictioni.e. an increase in muscle mass and/or strength.

A composition of the present disclosure can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the disclosureinclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrastemal injection andinfusion. In one embodiment the antibody is administered intravenously.In another embodiment the antibody is administered subcutaneously.

Alternatively, an antibody of the disclosure can be administered by anonparenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g. Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe disclosure can be administered with a needleless hypodermicinjection device, such as the devices shown in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.Examples of well known implants and modules useful in the presentdisclosure include: U.S. Pat. No. 4,487,603, which shows an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which shows a therapeutic device for administeringmedicants through the skin; U.S. Pat. No. 4,447,233, which shows amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which shows a variable flow implantableinfusion apparatus for continuous drug delivery; U.S. Pat. No.4,439,196, which shows an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which shows anosmotic drug delivery system. Many other such implants, deliverysystems, and modules are known to those skilled in the art and includethose made by MicroCHIPS (Bedford, Mass.).

In certain embodiments, the human monoclonal antibodies of thedisclosure can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the disclosurecross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g. U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g. V. V. Ranade,1989 J. Clin Pharmacol. 29:685). Example targeting moieties includefolate or biotin (see, e.g. U.S. Pat. No. 5,416,016); mannosides(Umezawa et al., 1988 Biochem. Biophys. Res. Commun. 153:1038);antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M. Owais etal., 1995 Antimicrob. Agents Chernother. 39:180); surfactant protein Areceptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120 (Schreieret al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen; M. L.Laukkanen, 1994 FEBSLett. 346:123; J. J. Killion; I. J. Fidler, 1994Imrnunomethods 4:273.

Uses and Methods of the Antibodies

The antibodies of the present disclosure have in vitro and in vivodiagnostic and therapeutic utilities. For example, these molecules canbe administered to cells in culture, e.g. in vitro or ex vivo, or in asubject, e.g. in vivo, to treat, prevent or diagnose a variety ofdisorders. Thus the antibodies may be used in both the treatment ofdisease, prophylaxis and for delaying the onset of disease symptoms. Theterm “subject” as used herein is intended to include human and non-humananimals. Non-human animals include all vertebrates, e.g. mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles.

The disclosure provides a method of treating a patient suffering from apathological disorder (such as a muscle wasting disease or disorder)comprising administering a therapeutically effective amount of ananti-ActRIIB antibody.

The disclosure also provides an anti-ActRIIB antibody for use intherapy.

The disclosure also provides use of an anti-ActRIIB antibody in themanufacture of a medicament for the treatment of a pathologicaldisorder.

The methods are particularly suitable for treating, preventing,ameliorating or diagnosing pathological disorders.

As used herein, a “pathological disorder” includes, but is not limitedto, musculoskeletal diseases or disorders, such as muscle atrophy. Thereare many causes of muscle atrophy, including as a result of treatmentwith a glucocorticoid such as cortisol, dexamethasone, betamethasone,prednisone, methylprednisolone, or prednisolone. The muscle atrophy canalso be a result of denervation due to nerve trauma or a result ofdegenerative, metabolic, or inflammatory neuropathy (e.g.,Guillian-Barré syndrome, peripheral neuropathy, or exposure toenvironmental toxins or drugs).

In addition, the muscle atrophy can be a result of myopathy, such asmyotonia; a congential myopathy, including nemalene myopathy,multi/minicore myopathy and myotubular (centronuclear) myopathy;mitochondrial myopathy; familial periodic paralysis; inflammatorymyopathy; metabolic myopathy, such as caused by a glycogen or lipidstorage disease; dermatomyositisis; polymyositis; inclusion bodymyositis; myositis ossificans; rhabdomyolysis and myoglobinurias.

The myopathy may be caused by a muscular dystrophy syndrome, such asDuchenne, Becker, myotonic, fascioscapulohumeral, Emery-Dreifuss,oculopharyngeal, scapulohumeral, limb girdle, Fukuyama, a congenitalmuscular dystrophy, or hereditary distal myopathy. The musculoskeletaldisease can also be osteoporosis, a bone fracture, short stature, ordwarfism.

In addition, the muscle atrophy can be a result of an adult motor neurondisease, infantile spinal muscular atrophy, amyotrophic lateralsclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathywith multifocal conductor block, paralysis due to stroke or spinal cordinjury, skeletal immobilization due to trauma, prolonged bed rest,voluntary inactivity, involuntary inactivity, metabolic stress ornutritional insufficiency, cancer, AIDS, fasting, a thyroid glanddisorder, diabetes, benign congenital hypotonia, central core disease,burn injury, chronic obstructive pulmonary disease, liver diseases(examples such as fibrosis, cirrhosis), sepsis, renal failure,congestive heart failure, ageing, space travel or time spent in a zerogravity environment.

Examples of age-related conditions that may be treated include,sarcopenia, skin atrophy, muscle wasting, brain atrophy,atherosclerosis, arteriosclerosis, pulmonary emphysema, osteoporosis,osteoarthritis, immunologic incompetence, high blood pressure, dementia,Huntington's disease, Alzheimer's disease, cataracts, age-relatedmacular degeneration, prostate cancer, stroke, diminished lifeexpectancy, frailty, memory loss, wrinkles, impaired kidney function,and age-related hearing loss; metabolic disorders, including Type IIDiabetes, Metabolic Syndrome, hyperglycemia, and obesity. Of course,patients may simultaneously suffer from one or more of these conditions,for example, sarcopenia and pulmonary emphysema, or sarcopenia andimpaired kidney function.

Other conditions that are considered to be “pathological disorders” asrecited herein include acute and/or chronic renal disease or failure,liver fibrosis or cirrhosis, cancer such as breast cancer, Parkinson'sDisease; conditions associated with neuronal death, such as ALS, brainatrophy, or dementia and anemia.

Further conditions include cachexia, cachexia associated with arheumatoid arthritis and cachexia associated with cancer.

To date, very few reliable or effective therapies have been developed totreat these disorders.

Based on reported evidence of a role of activins binding to ActRIIBamongst other receptors (Werner and Alzheimer, Cytokine Growth FactorsRev 2006, 17(3):157-171), in contributing to liver, kidney and pulmonaryfibrosis and of a role for myostatin, activins, or ActRIIB in cancers(Tsuchida et al, Endo J, 2008) the “pathological disorders” recitedherein include liver, kidney and pulmonary fibrosis and cancersexamplified by but not restricted to rhabdomyosarcomas, bone-lossinducing cancers, hepatocellular carcinomas, gastrointestinal cancers.

The prevention may be complete, e.g., the total absence of anage-related condition or metabolic disorder. The prevention may also bepartial, such that the likelihood of the occurrence of the age-relatedcondition or metabolic disorder in a subject is less likely to occurthan had the subject not received an antibody of the present disclosure.

An age-related condition as referred to herein may begin at the age of50 years or older (i.e. 60, 70, 80 or older).

In one embodiment, a patient may be pre-treated with an anti-ActRIIBantibody prior to an anticipated period of enforced rest/inactivity.Such a period may occur when a patient is admitted to hospital, forexample for surgery to the hip or leg. The inactivity may be localised,such as by casting of a broken limb or joint, or by administration of aparalytic agent.

In one embodiment, the patient being treated has a fracture to a limb(i.e. leg or arm) or joint (i.e. knee or hip). Thus, in one embodiment,the patient being treated has a fracture to one or more of the humerus,radius, ulnar, a carpal, a metacarpal, the clavical, scapular, femur, oscoxae, patella, tibia, fibula, talus, calcaneus, a tarsal, a metatarsal,the ischium or the ileum. In another embodiment, the patient beingtreated has undergone, or will undergo surgery on one or more of thefollowing joints: knee, hip, ankle, shoulder, elbow. Such surgeryincludes hip replacement and knee replacement.

Atrophy due to immobilisation may occur quickly, but normally occursslowly. Therefore, in one embodiment, the patient, joint or limb hasbeen immobilised, or will be immobilised, for 2 weeks or longer (i.e. 3weeks, 4 weeks, 6 weeks, 8 weeks or longer). In one embodiment, thepatient, joint or limb has been immobilised, or will be immobilised, for1-8 weeks, 2-6 weeks or 3-5 weeks.

In a further embodiment, the patient may be one who has not responded toprevious bone anabolic treatments. For example, the patient may not haveresponded to treatment with IGF-1, IGF-2 or variants of IGF-1 or IGF-2,an anti-myostatin antibody, a myostatin propeptide, a myostatin decoyprotein that binds ActRIIB but does not activate it, a beta 2 agonist, aGhrelin agonist, a SARM, GH agonists/mimetics or follistatin. A simpleway of measuring a patient's response to treatment may be timing howlong it takes for a patient to climb a known height of stairs andcomparing the results both before and after treatment.

The antibodies of the disclosure may be administered as the sole activeagent or in conjunction with, e.g. as an adjuvant to or in combinationto, other drugs e.g. IGF-1, IGF-2 or variants of IGF-1 or IGF-2, ananti-myostatin antibody, a myostatin propeptide, a myostatin decoyprotein that binds ActRIIB but does not activate it, a beta 2 agonist, aGhrelin agonist, a SARM, GH agonists/mimetics or follistatin. Forexample, the antibodies of the disclosure may be used in combinationwith an IGF-1 mimetic as disclosed in WO2007/146689.

In accordance with the foregoing the present disclosure provides in ayet further aspect:

A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount ofan ActRIIB antagonist, e.g. an antibody of the disclosure, and at leastone second drug substance, said second drug substance being IGF-1, IGF-2or variants of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatinpropeptide, a myostatin decoy protein that binds ActRIIB but does notactivate it, a beta 2 agonist, a Ghrelin agonist, a SARM, GHagonists/mimetics or follistatin.

The disclosure further provides a therapeutic combination, e.g. a kit,comprising of a therapeutically effective amount of a) an ActRIIBantagonist, e.g. an antibody of the disclosure, and b) at least onesecond substance selected from an IGF-1, IGF-2 or variants of IGF-1 orIGF-2, an anti-myostatin antibody, a myostatin propeptide, a myostatindecoy protein that binds ActRIIB but does not activate it, a beta 2agonist, a Ghrelin agonist, a SARM, GH agonists/mimetics or follistatin,e.g. as indicated above. The kit may further comprise instructions forits administration.

Where the antibodies of the disclosure are administered in conjunctionwith another active agent, dosages of the co-administered combinationcompound will of course vary depending on the type of co-drug employed,on the specific drug employed, on the condition being treated and soforth.

In another embodiment, the antibodies of the disclosure are administeredonly to a patient population which is selected among patients sufferingfrom muscle atrophy. In another embodiment, the antibodies of thedisclosure are administered to patient populations suffering fromskeletal muscle atrophy. In another embodiment, the antibodies of thedisclosure are administered only to a patient population which isselected among a group of patients which respond to anti-ActRIIBtreatment. Biomarkers that identify patients that have an increasedlikelihood of responding to anti-ActRIIB treatment may be any of thefollowing without being limited to these: high levels of serummyostatin, GDF-11 or activins compared to a control patient.

In one embodiment, the antibodies of the disclosure can be used todetect levels of ActRIIB, or levels of cells that contain ActRIIB. Thiscan be achieved, for example, by contacting a sample (such as an invitro sample) and a control sample with the anti-ActRIIB antibody underconditions that allow for the formation of a complex between theantibody and ActRIIB. Any complexes formed between the antibody andActRIIB are detected and compared in the sample and the control. Forexample, standard detection methods, well known in the art, such asELISA and flow cytometic assays, can be performed using the compositionsof the disclosure.

Accordingly, in one aspect, the disclosure further provides methods fordetecting the presence of ActRIIB (e.g. human ActRIIB) in a sample, ormeasuring the amount of ActRIIB, comprising contacting the sample, and acontrol sample, with an antibody of the disclosure, or an antigenbinding region thereof, which specifically binds to ActRIIB, underconditions that allow for formation of a complex between the antibody orportion thereof and ActRIIB. The formation of a complex is thendetected, wherein a difference in complex formation between the samplecompared to the control sample is indicative of the presence of ActRIIBin the sample.

Also within the scope of the disclosure are kits consisting of thecompositions (e.g. antibodies, human antibodies and bispecificmolecules) of the disclosure and instructions for use. The kit canfurther contain at least one additional reagent, or one or moreadditional antibodies of the disclosure (e.g. an antibody having acomplementary activity which binds to an epitope on the target antigendistinct from the first antibody). Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit. The kit may further comprisetools for diagnosing whether a patient belongs to a group that willresponse to an anti-ActRIIB antibody treatment, as defined above. Suchkits may comprise an antibody of the disclosure in lyophilised form, adiluent and instructions for use.

The disclosure having been fully described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting.

General

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y. The term “about” in relation to anumerical value x means, for example, x±10%.

DESCRIPTION OF THE FIGURES

FIG. 1 shows EC50 determination of MOR07079 by FACS titration onparental and ActRIIB transfected HEK293T/17 cell lines.

FIG. 2 shows inhibition of myostatin-induced luciferase expression in areporter gene assay by multiples of anti-ActRIIB Fabs at 2, 10 and 50μg/ml.

FIGS. 3A and 3B show IC50 determination of Fabs in myostatin-inducedluciferase reporter gene assay.

FIG. 4 shows antibody binding to primary human skeletal muscle cells.

FIGS. 5A and 5B show IC50 determination of IgG in myostatin-inducedinhibition of skeletal muscle differentiation assay.

FIGS. 6A, 6B, 6C, 6D and 6E show mouse study: in vivo efficacy study innaive animals −6 weeks treatment with MOR08159 or MOR08213 at 10 mg/kgincreased body and muscle weight. Changes are shown for (A) body weight,(B) Tibialis, (C) Gastrocnemius with plantaris, (D) Quadriceps and (E)Pectoralis.

FIGS. 7A, 7B, 7C, 7D and 7E show mouse study: dose response in vivoefficacy study in naive animals −6 weeks treatment with MOR08213 at 25,5, 1 mg/kg, dose-dependently increases body and muscle weight. Changesare shown for (A) body weight, (B) Tibialis, (C) Gastrocnemius withplantaris, (D) Quadriceps and (E) Pectoralis.

FIG. 8 shows a FACS output demonstrating cross-blocking between MOR08159in the presence of MOR08213 (bold dashed) and MOR08159 alone (boldblack), compared to isotype control alone (black) or isotype control inthe presence of MOR08213 (dashed).

FIG. 9 shows an overview of ActRIIB residues (SEQ ID NO:181) to whichMOR08159 binds, using various epitope determination techniques.

MODES FOR CARRYING OUT PARTICULAR METHODS Functional Assays ReporterGene Assay (Rga) Cultivation of HEK293T/17 Cell Lines

Parental HEK293T/17 cells are maintained in DMEM containing 10% FBS, 2mM L-glutamine, penicillin (50 IE/ml), and streptomycin (50 μg/ml).Cells are grown in an incubator at 37° C. and 5% CO₂ and subculturedevery 3-4 days. Cells are detached using Accutase™ and then transferredinto a new flask containing fresh medium.

HEK293T/17 cells stably transfected with CAGA-12 luc are cultured asdescribed above for parental HEK293T/17 cells but cell growth medium issupplemented with 4 mM L-glutamine and 3 μg/ml blasticidin in additionto FBS, penicillin and streptomycin.

Myostatin-Induced Luciferase Reporter Gene Assay

To determine the capacity of anti-ActRIIB antibodies to inhibitmyostatin-induced signaling, a reporter gene assay using the stablereporter cell line HEK293T/17 CAGA-12 luc is performed. The CAGA-12luciferase reporter construct carries the luciferase gene downstream ofa minimal promoter and multiple CAGA boxes which are specific forphosphorylated Smad-2 and Smad-3. Addition of purified myostatin (butalso of GDF-11, activin or TGFβ) induces Smad phosphorylation and thusbinding to the CAGA-12 reporter and leads to luciferase gene expression.

At 90% confluency of HEK293T/17 CAGA-12 luc cells, cells are detached asdescribed and diluted in culture medium to a concentration of 2.5×10⁵cells/ml. Subsequently, 100 μl cells per well are seeded intoflat-bottomed 96-well plates and incubated at 37° C. and 5% CO₂overnight.

The next day, the antibodies (Fab or IgG) and the recombinant humanActRIIB/Fc, which served as the positive control, are diluted in PBS tothe desired concentrations. 20 μl of the antibody solutions are added tothe seeded wells of the previous day and the cells cultivated for 1 hrto allow binding of the antibodies. Finally, 50 ng/ml myostatin is addedto the wells and the cells further cultivated over night.

The next morning, 120 μl Bright-Glo™ luciferase reagent (Promega) isadded to each well. After 2 min incubation time, the luminescence isread in a luminometer. The half maximal inhibitory concentration (IC50values) is calculated after full titration of the respective antibodies.

Specificity ELISAs

The specificity of anti-ActRIIB Fab antibodies to human ActRIIB andcrossreactivity to human ActRIIA and mouse ActRIIB is evaluated in anELISA setting. Additionally, binding to related receptors(counter-targets: human TGF-βRII/Fc (R&D systems), mouse TGF-βRI(ALK-5)/Fc (R&D systems), human Activin RIB (ALK-4)/Fc (R&D systems)) isdetermined. For this, 5 μg/ml (if not stated otherwise) of therecombinant proteins diluted in PBS are added to a black 96-wellflat-bottom MaxiSorp plate and incubated over night at 4° C. forcoating.

The next morning, the plates are washed with TBST and blocked withMTBST. After washing the plates several times, 5 μg/ml anti-ActRIIB Fabare added and incubated for 2.5 hrs. Subsequently antigen bound Fabs aredetected by incubation with alkaline phosphatase conjugatedgoat-anti-human IgG Fab-specific, followed by addition of AttoPhosfluorescence substrate. Fluorescence emission at 535 nm is recorded withexcitation at 430 nm in a TECAN Spectrafluor plate reader.

ActRIIB/Fc-Myostatin Binding Interaction ELISA

To assess whether the inhibitory Fabs act via blocking the myostatinbinding site of human ActRIIB a hActRIIB/Fc-myostatin interaction ELISAis performed. For this, recombinant myostatin is diluted to 5 μg/ml inPBS and coated onto a black 96-well flat-bottom Maxisorp plate. The nextmorning the wells are blocked with MTBST. Meanwhile 50 μg/mlanti-ActRIIB Fabs are pre-incubated with 10 μg/ml ActRIIB/Fc in TBST for1.5 hrs at room temperature and finally added to the coated and blockedwells (1.5 hr at room temperature). After washing with TBST buffer,detection of bound ActRIIB/Fc is performed using an unlabelled mouseanti-human Ig Fc-specific antibody and a POD-labelled sheep anti-mouseIgG detection antibody. After washing the wells several times with TBSTbuffer Quanta Blu™ Fluorogenic Peroxidase substrate is added. Thefluorescence is read in a GENiosPro reader (excitation 320 nm, emission430 nm).

Binding to Cells Cells

Stable human ActRIIA- and human ActRIIB-transfected HEK293T/17 cells,generated using HEK293T/17 cells (ATCC) transfected with linearizedpEGFP (Clontech)-ActRIIB(ECD) or -ActRIIA(ECD) and pPGK-puro (AddGene)using FuGENE6 (Roche), are maintained in DMEM containing 10% FBS, 2 mML-glutamine, penicillin (50 IE/ml), streptomycin (50 μg/ml) andpuromycin (2 μg/ml). Cells are grown in an incubator at 37° C. and 5%CO₂ and subcultured every 3-4 days. Cells are detached using Accutaseand then transferred into a new flask containing fresh media.

Human skeletal muscle cells (huSkMC) (Cambrex) are harvested at aconfluency of about 70-90%. For those cells, culture medium, growthmedium (GM) consisting of skeletal muscle basal medium (skBM; Lonza)supplemented with 20% FCS (Amimed), is aspirated, and the cells arewashed with HEPES-BSS and incubated with Trypsin/EDTA. After the cellsare detached, trypsin is neutralized with trypsin neutralizing solution.Cells are centrifuged at 220×g for 5 min and the pellet is resuspendedin Skeletal Muscle Growth Media. Cells are then used for experiments orseeded for subculturing at a cell density of ˜3500 cell/cm². Cells aregrown in an incubator at 37° C. and 5% CO₂ and subcultured every 5-6days.

FACS Titration on hActRIIB- and hActRIIA-Expressing Cells

The half maximal effective concentration (EC50) of the anti-ActRIIBantibodies is determined via binding to cellular hActRIIA and hActRIIBby FACS.

For this, serial dilutions of anti-ActRIIB Fab or IgG are incubated with1×10⁴ hActRIIA-transfected, hActRIIB-transfected or parental HEK293T/17cells per well for 1 h at 4° C. After several washing steps cell-boundFabs or IgGs are detected with phycoerythrin-conjugated goat anti-humanIgG (H+L) secondary antibody. After one hour incubation at 4° C., thecells are washed again and resuspended in FACS buffer and fluorescenceintensity of the cells is determined in a FACSArray™ instrument.

Binding to Primary Human Skeletal Muscle Cells

Anti-ActRIIB Fab or IgG as well as isotype control Fab or IgG (10 μg)are incubated with 10⁵ huSkMC in FACS buffer (PBS, 2% FCS, 1 mM EDTA)per tube for 1 h at 4° C. After washing steps, cell-bound Fabs or IgGsare detected with phycoerythrin-conjugated goat anti-human IgG (H+L)secondary antibody which had been diluted 1:200 in FACS buffer. Afterone hour incubation at 4° C. on a shaker, the cells are washed again andresuspended in FACS buffer and fluorescence intensity of the cellsdetermined in the FACSCaliber™ instrument.

Affinity Determination Affinity Determination of Selected Anti-HumanActRIIB Fabs Using Surface Plasmon Resonance (Biacore)

For direct antigen immobilisation standard EDC-NHS amine couplingchemistry is used. CMS chips (Biacore, Sweden) are coated withapproximately 6000 RU human- or mouse-ActRIIB/Fc, or approximately 1500RU human-ActRIIA/Fc (according to the activity of the antigens) in 10 mMacetate buffer, pH 4.5. For the reference flow cell, a respective amountof HSA is used. Regeneration is done with 5 μl 10 mM Glycine/HCl bufferpH1.5.

Alternatively, the antigens are not immobilized directly, but capturedon a CMS chip, which is modified with an anti-human-Fc antibody (Fccapture kit, GE Healthcare/Biacore). On the reference flow cell, captureantibody is immobilized, but no antigen captured. Regeneration isachieved using 2 injections of 5 μL 3M MgCl₂.

Kinetic measurements are done in Dulbecco's PBS at a flow rate of 20μl/min using a serial dilution row of Fab samples. The Fabconcentrations ranged from 15.6 to 500 nM. Injection time for eachconcentration is 1 min. The dissociation time is set to at least 2 min(or more, according to determined affinity). A blank injection ofrunning buffer is used for double referencing. All sensorgrams arefitted globally using BIA evaluation software 3.2 (Biacore, Sweden).

CK Assay

Differentiation is initiated 24 hours after seeding by changing cellsfrom GM to serum-free differentiation medium consisting of skeletalmuscle basal medium (skBM). Cells are differentiated for 3 days in theabsence and presence of myostatin (R&D systems) or other TGF-b proteinsand tested antibodies at given concentrations. Cells are washed with PBSand then lysed with Reporter lysis buffer (Promega) and stored tillmeasurement at −80° C. CK activity is measured using the CK (IFCC)reagent (Thermo Electron). The CK reagent is prepared according to themanufacturers instructions. Cell lysates are adjusted to roomtemperature, CK reagent is added and absorbance is immediately read at340 nm for 20 min, reading interval 1 min. CK standard curves arefreshly prepared using CK from rabbit muscle (Roche Diagnostics).Protein content is determined using BCA kit.

Animal Models

Nine-week-old female CB17/ICR-Prkdc^(scid)/Crl mice (n=10 per group,Charles River, Germany) are randomized with body weight and then treatedintraperitoneally with anti-human ActRIIB antibodies (MOR8159, MOR8213)or IgG control antibody at a dose of 10 mg/kg (Study 1; comparisonstudy), or with MOR8213 at doses of 25, 5, or 1 mg/kg (Study 2; doseresponse study) on day 0, 3, 7, 14, 21, 28 and 35 (once weekly with day3). Body weights are determined two times per week. Six weeks (42 days)after administration, mice are euthanized with CO₂. Tibialis,gastrocnemius with plantaris, quadriceps and pectoralis are collectedand weighed.

Treatment Protocol

Control antibody: anti-chicken lysozyme-hIgG,Concentration: 2 mg/mL (study 1), 5 mg/mL (study 2), application volume:5 mL/kgVehicle: 50 mM Citrate, 140 mM NaCl or PBS anti-human ActRIIBantibodies: anti-ActRIIB-MOR8159 and MOR8213, hIgG,Concentration: 2 mg/mL (study 1), 5 mg/mL (study 2), 1 mg/mL (study 2),0.2 mg/mL (study 2), application volume: 5 mL/kg

Vehicle: 50 mM Citrate, 140 mM NaCl Treatment Groups: Study 1;Comparison of MOR08159 and MOR08213

1 IgG control, i.p. (anti-chicken lysozyme-IgG), 10 mg/kg2 anti-ActRIIB-MOR8159, i.p., 10 mg/kg3 anti-ActRIIB-MOR8213, i.p, 10 mg/kgStudy 2; dose response of MOR082131 IgG control, i.p. (anti-chicken lysozyme IgG), 25 mg/kg2 anti-ActRIIB-MOR8213, i.p., 25 mg/kg3 anti-ActRIIB-MOR8213, i.p., 5 mg/kg4 anti-ActRIIB-MOR8213, i.p., 1 mg/kg

Maintenance Conditions

Animals are housed in groups of four to five animals at 25° C. with a12:12 h light-dark cycle. They are fed a standard laboratory dietcontaining 18.2% protein and 3.0% fat with energy of 15.8 MJ/kg (NAFAG3890, Kliba). Food and water are provided ad libitum. Animalexperimentation is carried out according to the regulations effective inthe Canton of Basel-City, Switzerland.

Methods Statistical Analysis

Results are expressed as mean+/−SEM. Statistical analysis is carried outusing Dunnett's multiple comparison test following one-way analysis ofvariance. Treatment (anti-ActRIIB antibodies MOR8159 and MOR8213 aretested for difference to control (control antibody) and differences areconsidered to be significant when the probability value is <0.05: *:P<0.05, **: P<0.01, NS: no significance versus IgG control. Statisticalanalyses are performed by GraphPad Prism version 5.0 (GraphPad Software,Inc). Body weight are calculated by subtracting body weight at day 0,and muscle weight is normalized by body weight at day 0 (initial bodyweight).

Pannings, Antibody Identification and Characterization

Therapeutic antibodies against human ActRIIB protein are generated byselection of clones having high binding affinities, using as the sourceof antibody variant proteins a commercially available phage displaylibrary, the MorphoSys HuCAL GOLD® library.

HuCAL GOLD® library is a Fab library (Knappik et al., 2000) in which allsix CDRs are diversified by appropriate methods, and which employs theCysDisplay technology for linking the Fab to the phage surface(WO01/05950).

HuCAL GOLD® phagemid library (Rothe et al., 2008) is used to selectspecific Fab antibody fragments.

Selection by Panning of ActRIIB-Specific Antibodies from the Library

For the selection of antibodies recognizing human ActRIIB severalpanning strategies are applied.

In summary, HuCAL GOLD® antibody-phages are divided into several poolscomprising different VH master genes.

These pools are individually subjected to differential cell panningswhereby selection rounds on transiently human ActRIIB transfected cellsalternated with selection rounds on recombinant human ActRIIB/Fcprotein.

i. Whole Cell Panning

For the pannings, phage particles diluted in PBS are mixed with an equalvolume of PBS/BSA and blocked. In parallel, also in pre-blocked tubes,1×10⁷ of the respective hActRIIB expressing cells per phage pool areresuspended in PBS/3% FCS/0.04% NaN₃ and blocked for one hour at 4° C.on a shaker. The blocked cells are spun down, resuspended in thepre-blocked phage particles and incubated for three hours. In themeantime, 1×10⁷ hActRIIB knock-down cells per phage pool are prepared.

The phage-cell complexes are washed in PBS/BSA, followed by washing inPBS. Elution of phage particles from the hActRIIB expressing cells isperformed by acidic elution with glycine buffer, pH 2.2. Aftercentrifugation, the eluate is neutralized by adding unbuffered Tris.

After infection and subsequent centrifugation, the bacterial pellets areresuspended in 2×YT medium, plated onto LB/CAM/Glc agar plates andincubated overnight at 37° C. The next morning, the colonies are scrapedoff the plates and the phages are rescued and amplified.

ii. Solid Phase Panning

For solid phase panning recombinant human ActRIIB/Fc is coated onto aMaxiSorp plate at 4° C. over night. After washing with PBS the coatedwells are blocked with 5% MPBST.

Prior to the selections, HuCAL GOLD® phage are pre-adsorbed in blockingbuffer. The blocked phage are added to the coated antigen and incubatedfor 2 hrs at room temperature. Unspecific phage are washed off with PBSTand PBS. Bound phage are eluted by addition of 20 mM DTT. The eluatesare used for infection of an E. coli TG-1 culture. After infection, thebacteria are plated onto LB/CAM/Glc agar plates and incubated overnightat 37° C. The next morning, the colonies are scraped off the plates andthe phage are rescued and amplified.

The most successful panning approach proved to be differentialcell/protein pannings with first panning round on ActRIIB transfectedHEK293T/17 cells followed by selection round on recombinant humanActRIIB/Fc and again on transfected cells.

Selected Fabs are analyzed for binding to parental or rhActRIIBtransfected HEK293 cells.

MOR07079 Fab binds in various embodiments to ActRIIB-transfected cellswith an EC50 of 20 nM (FIG. 1). In a myostatin binding inhibition ELISA,MOR07079 Fab showed inhibitory activity and blocked rhActRIIB/Fc bindingto myostatin. Strong myostatin binding inhibition in ELISA is reflectedby myostatin inhibition in the reporter gene assay using HEK293-CAGA12for MOR07079. Using the specificity ELISA, MOR07079 is shown to bindspecifically to human and murine ActRIIB and not to the unrelatedTGFβRII, ALK4 and ALK5 receptors. MOR7079 is also shown to bind invarious embodiments to ActRIIB compared to ActRIIA.

Production of HuCAL® Immunoglobulins

i. Conversion of Fabs into the IgG Format

In order to express full length immunoglobulin (Ig), the variable domainfragments of heavy (VH) and light chains (VL) are subcloned from thepMORPH®X9_FH Fab expression vectors into the pMORPH®2_h_Ig vector seriesfor human IgG2. Selected clones are also converted into the silentIgG1LALA format in which leucines at positions 234 and 235 are mutatedto alanines to abrogate FcRγ binding and attenuate effector functions.

Appropriate restriction enzymes (Knappik et al., 2000) are used forsubcloning of the VH and VL domain fragments into pMORPH®2_h_IgG2,pMORPH®2_h_IgG1LALA, pMORPH®2_h_Igκ, and pMORPH®2_h_Igλ2.

All DNA preparations are subjected to sequence analysis beforetransfection into HKB11 cells.

ii. Transient Expression and Purification of Human IgG

Eukaryotic HKB11 cells are transfected with IgG heavy and light chainexpression vector DNA. Cell culture supernatant is harvested at 3 or 7days post transfection and subjected to standard protein A affinitychromatography. If not otherwise stated, buffer exchange is performed to1× Dulbecco's PBS (pH 7.2) and samples are sterile filtered (0.2 μm).

CDR-L3 and CDR-H2 Maturation Libraries

To increase affinity and biological activity of selected antibodyfragments, CDR-L3 and CDR-H2 regions are optimized in parallel bycassette mutagenesis using trinucleotide directed mutagenesis (Virnekaset al., 1994, Nucleic Acids Res. 22:5600-5607), whereby the frameworkregions are kept constant (Nagy et al., 2002, Nature Medicine,8:801-807). Prior to cloning of the maturation libraries, all parentalFab fragments are transferred from the expression vector pMORPH®X9 intothe CysDisplay™ maturation vector pMORPH® 25 via the XbaI/EcoRIrestriction sites. This vector provides the phage protein pIII fusedN-terminally to a cysteine residue as well as a C-terminal cysteinefused to the Fd antibody chain and thus allows disulfide-linked displayof the respective Fab fragments on the phage surface.

For generation of the CDR-H2 libraries the CDR-H2 region of eachparental Fab is excised and replaced by the highly diversified CDR-H2maturation cassette.

In parallel, the CDR-L3 region of the parental clones is replaced by adiversified CDR-L3 maturation cassette.

The sizes of the maturation libraries ranged from 4×10⁵ to 1×10⁸ clones.The vector background is below 1% in all cases. The quality control bysequencing of single clones revealed a high quality of each library.

For each CDR-L3 and CDR-H2 maturation library, antibody-displaying phageare prepared and phage titers determined by spot titration.

Panning Strategies for Affinity Maturation

The antibody-displaying phages from the following maturation librariesare subjected to separate pannings and screenings:

Lead 1: MOR07079 (L-CDR3 maturation)Lead 1: MOR07079 (H-CDR2 maturation)

Maturation pannings using the respective antibodies are performed onbiotinylated hActRIIB/Fc and on huSkMC.

Either 2×10¹⁰ or 1×10¹¹ phages per subcode, rescued from the newlygenerated maturation libraries are used for the first selection rounds.

Several differential pannings are performed whereby selection rounds onrecombinant biotinylated hActRIIB/Fc alternated with a selection roundon huSkMC.

For the first and third round of the solution panning biotinylatedrecombinant hActRIIB/Fc is captured onto Streptavidin-coated Dynabeads.The following protocol is applied: for each phage pool, Streptavidinbeads are washed with PBS and resuspended in blocking buffer. Phageparticles diluted in PBS are mixed blocking buffer containing 0.1%Tween20 and kept on a rotating wheel. Preclearing of phage particles forremoval of Streptavidin- or bead-binding phages is performed twice: Perphage pool blocked Streptavidin beads are added to the blocked phageparticles and incubated on a rotating wheel. After separation of thebeads via a magnetic device the phage supernatant is transferred to afresh, pre-blocked reaction tube and pre-adsorption is repeated.

After the blocking procedure, the biotinylated hActRIIB/Fc antigen isadded to the precleared and blocked phage particles and incubated on arotating wheel. The phage-antigen complexes are captured using blockedstreptavidin beads, added to the phage panning pools and incubatedfurther. Phage particles bound to the streptavidin beads are collected.Beads are then washed with PBST and PBS. Elution of phage particles fromthe streptavidin beads is performed by addition of 20 mM DTT. The eluateis collected and used for infection of an E. coli TG-1 culture grown toan OD_(600 nm) of 0.6-0.8.

After infection and subsequent centrifugation, the bacterial pellets areresuspended in 2×YT medium, plated onto LB/CAM/Glc agar plates andincubated overnight at 37° C. The next morning, the colonies are scrapedoff the plates and the phages are rescued and amplified mainly asdescribed (Krebs et al., 2001) with the exception that helper phageinfected cells are grown at 22° C. over night in medium containing 0.25mM IPTG. The third rounds of the solution pannings on biotinylatedhActRIIB/Fc are performed according to the protocol of the first roundexcept for decreasing amounts of antigen used and increased stringencyof the washing conditions.

For the second round of panning (on huSkMC expressing endogenoushActRIIB), phage particles diluted in PBS are mixed with an equal volumeof PBS/BSA and blocked. In parallel, for each subcode 9×10⁵ huSkMC areblocked with PBS/FCS/0.02% NaN₃ at 4° C. The blocked cells are spundown, resuspended together with the pre-blocked phage particles andincubated further.

The phage-cell complexes are washed with PBS/BSA, followed by washing inPBS. Cells are centrifuged at 410×g for 2 min at 4° C. Acidic elution ofphage particles from the hActRIIB expressing huSkMC is performed by a 10min incubation step with glycine buffer, pH 2.2. After centrifugation,the eluate is neutralized by adding unbuffered Tris. The phagecontaining supernatant is used for infection of an E. coli TG-1 culturegrown to an OD_(600 nm) of 0.6-0.8.

After infection and subsequent centrifugation, the bacterial pellets areresuspended in 2×YT medium, plated onto LB/CAM/Glc agar plates andincubated overnight at 37° C. The next morning, the colonies are scrapedoff the plates and the phages are rescued and amplified mainly asdescribed (Krebs et al., 2001) with the exception that helper phageinfected cells are grown at 22° C. over night in medium containing 0.25mM IPTG.

The most successful panning approach which resulted in very potentbinders proved to be the differential panning with the first and thirdround performed on biotinylated ActRIIB/Fc and the second round onhuSkMC.

After sequencing, Fabs are selected for expression and purification, andthe most promising further characterized.

Most anti-ActRIIB antibodies showed binding to hActRIIB-transfectedHEK293T/17 cells with EC50 values in the single up to low double digitnanomolar range. Several Fabs could displace myostatin from ActRIIB/Fcin a myostatin binding inhibition ELISA, but amongst those only MOR08067displayed full inhibition of myostatin-induced activity in the reportergene assay (FIG. 2).

Summarized affinities of the most promising Fab to human and mouseActRIIB/Fc are listed in the table below (Table 1).

TABLE 1 Affinity data of anti-ActRIIB Fab-FH to ActRIIB antigens KDdetermination (Biacore) human ActRIIB- mouse ActRIIB- Fc KD Fc KD Fab[nM] [nM] MOR07079 51 62 MOR08047 23 22 MOR08062 15 17 MOR08067 <0.1<0.1 MOR08077 11 13 MOR08078 9 10

The Fab clone MOR08067 exhibited good inhibition in the myostatininduced RGA as well as binding to rhActRIIB transfected HEK293 cells.Affinity determination by Biacore revealed KD values to human and mouseActRIIB/Fc below 100 pM. MOR08067 and other candidates are selected forfurther optimization by a cross cloning approach, while MOR08067,containing a potential N-linked glycolsylation site is also subjected toa deglycosylation approach.

Optimization of antibodies derived from first affinity maturation

a) Deglycosylation of MOR08067

According to sequence analysis this antibody contained a potentialN-linked glycosylation site within the CDR-H2 of the heavy chain. Thissite is removed to yield MOR08156 and MOR08159. The characterization ofthese MOR08067-derivatives is described below.

b) Cross Cloning of Optimized Fabs

For a further functional improvement and removal of potential N-linkedglycosylation sites in CDR-H2s and/or CDR-L3s, the independentlyoptimized CDR-H2 and CDR-L3 regions from single affinity matured Fabsresulting from the first affinity maturation are combined while keepingeach family separate. Descendants of MOR07079 entered cross cloning.Roughly 200 bacterial lysates are tested in FACS affinity ranking onHEK293T/17/ActRIIB and the most promising Fab clones, MOR08144 andMOR08213 are expressed, and purified.

c) Characterization of Optimized Antibodies

In the following sections, the deglycosylated progenies of MOR08067(MOR08156, MOR08159) and the two cross clones derived from MOR08067(MOR08144 and MOR08213) are described in detail.

The ability of optimized Fabs to inhibit myostatin signaling in thereporter gene assay is determined, with all binders being able toinduce >95% inhibition at the highest concentration (FIG. 3A and FIG.3B).

In affinity determination experiments using Biacore, MOR08159 andMOR08213 are identified as highly potent binders to both human and mouseActRIIB (Table 2). It became obvious the increased affinity of maturedand optimized Fabs reflected increased potency in the myostatin-inducedreporter gene assay.

TABLE 2 Affinity data of anti-ActRIIB Fabs to ActRIIB antigens KDdetermination (Biacore) human ActRIIB- mouse ActRIIB- Fc KD Fc KD Fab[pM] [pM] MOR08159 3.8 3.1 MOR08213 13.2 13.5

IgG2 Conversion of Affinity Matured Fabs (1^(st) Maturation)

The most promising Fabs derived from the first affinity maturation areselected for IgG2 conversion.

IgG2 expression is performed by transient transfection of HKB11 cellsand the full length immunoglobulins are purified from the cell culturesupernatants.

Upon conversion to IgG, all candidates retained their ability todose-dependently inhibit myostatin-induced activity in the reporter geneassay (Table 3).

TABLE 3 IC50 determination of anti-ActRIIB IgGs in myostatin- inducedluciferase reporter gene assay IgG IC50 [nM] % inhibition MOR08067 2.5786.5 MOR08144 0.5 94.9 MOR08156 0.19 97.4 MOR08159 0.32 99 MOR08213 0.3298.6

MOR08159 and MOR08213 are tested for their ability to bind to humanprimary muscle myoblasts by FACS, and specific binding to those cells isreported, in line with low expression of ActRIIB on those cells (FIG.4).

MOR08159 (FIG. 5A) and MOR08213 (FIG. 5B) displayed ability to fullyreverse the myostatin-induced inhibition of primary skeletal myoblastsdifferentiation. Those antibodies also increased differentiation abovebasal level in the absence of exogenous myostatin, due to their abilityto neutralize endogenously produced ActRIIB ligands.

Second Affinity Maturation

Selection of Candidates for Second Affinity Maturation to furtherimprove the efficacy.

i. Construction of the CDR-L3 and CDR-H2 Maturation Libraries

To increase both affinity and biological activity of the selectedantibody fragments (e.g. MOR08067), CDR-L1 and CDR-H2 regions areoptimized by cassette mutagenesis using trinucleotide directedmutagenesis (Virnekas et al. [supra]), whereby the framework regions arekept constant (Nagy et al. [supra]). Prior to cloning of the maturationlibraries, all parental Fab fragments are transferred from theexpression vector pMORPH®X9 into the CysDisplay™ maturation vectorpMORPH®25 via the XbaI/EcoRI restriction sites.

The sizes of all maturation libraries yielded always a minimum of 1×10⁷independent clones.

The vector background is below 1% in all cases. Quality control bysequencing of single clones revealed a high quality of each library.

For each CDR-L1 and CDR-H2 maturation library, antibody-displayingphages are prepared and phage titers are determined by spot titration.

ii. Panning Strategies, Affinity Ranking and Screening for ImprovedAntibodies

Differential pannings for the second round of affinity maturationincluded parental HEK293T/17 cells and huSkMC expressing human ActRIIBendogeneously at low levels. Additionally, recombinant biotinylatedhActRIIB/Fc antigen is included in all panning strategies.

For ranking of the anti-ActRIIB Fabs, approximately 2700 bacteriallysates (˜88 clones for each panning subcode) are affinity ranked onrecombinant biotinylated hActRIIB/Fc antigen and membrane vesiclepreparations of hActRIIB-transfected HEK293T/17 cells in an MSD-basedmethod. Hits with high affinity ranking factor are sequenced.

In addition, randomly selected clones which did not show up as hits areevaluated in FACS affinity ranking. For this, bacterial lysates arescreened using parental HEK293T/17 and/or hActRIIB-transfectedHEK293T/17 cells. Cell-bound Fabs are detected withphycoerythrin-conjugated goat anti-human IgG (H+L) secondary antibody.The quantification of Fab expression in the lysates is performed inparallel.

All panning strategies yielded anti-ActRIIB specific antibodies.MOR08067 progenies could be identified after sequence analysis. Allbinders are matured in CDR-H2.

IgG2 Conversion and Characterization of IgG2 (2^(nd) Maturation)

Again, the most promising Fabs derived from the second affinitymaturation are selected for IgG2 conversion. IgG2 expression isperformed by transient transfection of HKB11 cells and the full lengthimmunoglobulins are purified from the cell culture supernatants.

All IgGs tested are able to fully reverse the myostatin-inducedinhibition of primary skeletal myoblasts differentiation (table 4).

TABLE 4 IC50 determination of anti-ActRIIB IgGs in myostatin-inducedinhibition of skeletal muscle differentiation assay CK assay IgG IC50[nM] MOR08159 1.89 MOR08213 1.7 MOR08806 0.52 MOR08807 5.02 MOR090321.02 MOR09058 2.3

We evaluated the ability of the anti-ActRIIB Ab to neutralize binding ofmyostatin as well as other TGFβ family ligands to ActRIIB on primaryhuman skeletal myoblasts. In the myoblast differentiation assay, weassessed the various ligands potential to inhibit differentiation in theabsence or presence of either MOR08159 or MOR08213.

TABLE 5 IC50 and Emax determination of various ligand-induced inhibitionof skeletal muscle differentiation assay in the presence or absence ofMOR08159/MOR08213 (10 μg/ml) no Ab MOR08159 MOR08213 TGFβ family IC₅₀E_(max) IC₅₀ E_(max) IC₅₀ E_(max) ligands (ng/ml) (% control) (ng/ml) (%control) (ng/ml) (% control) Myostatin  8.5 ± 0.6 25.7 ± 1.5 42.7 ± 5.828.9 ± 4.8 35.1 ± 5.1 35.3 ± 4.5 GDF-11  7.0 ± 1.2 23.2 ± 3.8 13.3 ± 0.922.1 ± 2.1 12.0 ± 1.0 27.1 ± 2.6 Activin A 14.7 ± 2.9 37.1 ± 3.9 34.7 ±9.0 61.9 ± 5.4 41.9 ± 3.4 57.2 ± 1.9 BMP-2 26.9 ± 2.6  2.6 ± 4.2 34.0 ±2.6  5.1 ± 3.4 32.3 ± 1.4  4.8 ± 1.9

Myostatin and GDF-11 are able to inhibit human myoblasts differentiationwith similar efficiencies and to similar extents. In the presence of asingle concentration of MOR08159 or MOR08213, myostatin and GDF-11 doseresponses are shifted in a parallel manner. Activin A is also able toinhibit differentiation, however in the presence of MOR08159 orMOR08213, we observed a non parallel shift accompanied by a change inEmax and potency. BMP-2 response is unaffected by the presence ofMOR08159 or MOR08213, suggesting that it does not occur via ActRIIBbinding.

Characterization of Anti ActRIIB Antibodies in In Vivo Murine Studies.

The ability of anti-ActRIIB antibodies to induce muscle hypertrophy isevaluated in SCID mice administered MOR08159 or MOR08213, 10 mg/kg i.p.weekly for 6 weeks (FIG. 6).

Both antibodies are able to induce a profound hypertrophy of allexamined muscles at study end. Significant increase in overallbodyweight of anti-ActRIIB antibody treated mice is detected as early asafter 1 week of treatment.

MOR08213 is able to induce a dose-dependent profound hypertrophy of allexamined muscles at 5 and 25 mg/kg while no significant changes arenoticed at 1 mg/kg dose (FIG. 7).

Cross Blocking Studies

Stable human ActRIIB-transfected HEK293T/17 cells are maintained in DMEMcontaining 10% FBS, 2 mM L-glutamine, penicillin (50 IE/ml),streptomycin (50 μg/ml) and puromycin (2 μg/ml). Cells are grown in anincubator at 37° C. and 5% CO₂ and subcultured every 3-4 days. Cells aredetached using Accutase and then transferred into a new flask containingfresh media.

The ability of the anti-ActRIIB antibodies to bind to the same epitopeof human ActRIIB is assessed by FACS using hActRIIB-expressing cells.

For this, an anti-ActRIIB IgG is incubated with 1×10⁵hActRIIB-transfected cells per well for 1 h at 4° C. After washing, adifferent biotinylated anti-ActRIIB IgG or a control biotinylated IgGare incubated at equimolar concentration to the first anti-ActRIIB IgGfor 1 h at 4° C. After washing, cell-bound biotonylated IgG are detectedwith streptavidin-APC (Biolegend). After one hour incubation at 4° C.,the cells are washed again and resuspended in FACS buffer andfluorescence intensity of the cells is determined in a FACSArrayinstrument.

MOR08159 and MOR08213 are tested for their ability to jointly bind tohuman ActRIIB-transfected cells by FACS, and specific binding ofMOR08159 alone (bold black) or in the presence of MOR08213 (bold dashed)is reported compared to isotype control (black) or isotype control inthe presence of MOR08213 (dashed) (FIG. 8).

In the presence of MOR08213, binding of MOR08159 is significantlyreduced suggesting that those two antibodies either bind to the samesites or to sites that may have some degree of overlap or that bindingof MOR08213 to a distinct, but nearby site, might sterically hinderbinding of MOR08159.

Epitope Mapping

Several complementary methods are used to determine the epitope to whichthe antibody MOR08159 binds. In this example, residue numbering is withreference to the full length ActRIIB amino acid sequence (SEQ ID NO:181).

Dot Blot

A dot blot analysis of the MOR08159 epitope is carried out. Native anddenatured (reduced and heat-denatured) ActRIIB is spotted on anitrocellulose membrane, probed with MOR08159, and detected with alabeled anti-human antibody. Only native ActRIIB, but not reduced andheat-denatured ActRIIB is detected. The results indicated that theepitope is a conformational epitope.

Mutational Studies

A library of the extracellular domain of ActRIIB (aa 21-120) isgenerated by error-prone PCR and the variants are expressed in theperiplasm of E. coli. The binding of about 30′000 (small fraction of thetheoretical library size) of those variants to MOR08159 is tested bycolony filter screening and western staining. Variants which showed onlyweak or no binding to MOR08159 are further confirmed by ELISA.Expression level (detected with an anti-Flag antibody) and MOR08159binding of the ActRIIB variants are compared to wild-type ActRIIB. Ifexpression is at least 75% of wild type and binding to MOR08159 is lessthan 25%, the mutation is rated to be involved in MOR08159 binding. Onlyvariants having a single point mutation, which is not obviouslystructure distorting (as e.g. the mutation of an S-S bridging cysteinewould be) are considered.

Most mutations which prevented MOR08159 binding are found in a stretchfrom position K75 to D81, which indicates that this region is importantfor antibody binding. Mutations at positions W78, D80 and D81 are foundto reduce MOR08159 binding significantly.

Cyclic Peptide Arrays

A collection of antigen derived cyclic peptides displayed on peptidemicroarrays are incubated with antibodies of interest. The determinationof peptide-antibody binding is performed by RepliTope-analysis where thepeptide microarray is incubated with the primary antibody followed by afluorescently labelled secondary antibody directed against the Fc-partof the primary one. After several washing steps the peptide microarrayswhere dried using a microarray centrifuge and scanned in a highresolution microarray scanning system with appropriate wavelengthsettings.

The microarray is composed of three subarrays, each displaying cyclicpeptides derived from ActRIIB (with Cys residue exchanged to Ser), whichare scanned (peptide scan format 15/12). As control experiment, oneincubation with unrelated antibody (ACE18543, isotope control) followedby fluorescently labelled secondary antibody (Cy-5 labelled anti-humanIgG) is performed to determine false positive signals. Additionally,incubation with target antibody, followed by fluorescently labelledsecondary antibody, is performed.

Antibody MOR08159 (ACE19819) is shown to recognise one epitope, which isfound in three of the tested peptides (nos. 18-20).

18 IELVKKGSWLDDFNS (SEQ ID NO: 183) 19    VKKGSWLDDFNSYDR(SEQ ID NO: 184) 20       GSWLDDFNSYDRQES (SEQ ID NO: 185)

The sequence common to these peptides to which MOR08159 is considered tobind is 76GCWLDDFNC84 (SEQ ID NO: 186).

A second region with weaker binding characteristics is also identifiedusing this method. This second region has the sequence49CEGEQDKRLHCYASW63 (SEQ ID NO: 187).

X-Ray Crystallography

Human ActRIIB aa20-120 and aa24-117 are expressed. In addition, MOR08159Fab and Fv regions are expressed and purified (all expression is carriedout in E. coli). Using these proteins, four protein complexes areprepared, purified and crystallised (MOR08159Fab-ActRIIB 20-120,MOR08159Fab-ActRIIB 24-117, MOR08159Fv-ActRIIB 20-120,MOR08159Fv-ActRIIB 24-117).

The x-ray structure of free MOR08159 Fab is resolved to 1.78 Åresolution. The x-ray structure of the Fv complex with ActRIIB-LBD isresolved to 3.35 Å resolution. Using the standard 3.9 Å distance cut-offto determine contact residues, it is confirmed that the sequence76GCWLDDFNC84 is an important region with dominant binding contributionfrom the 78WLDDFN83 sequence (SEQ ID NO: 188). In addition, interactionis also found with the peptide region 49CEGEQDKRLHCYASW63.

The results for the various epitope mapping experiments are summarisedin FIG. 9.

Confirmation of Affinity by SET

Serial dilutions of antigen (extracellular domain of ActRIIB or ActRIIA)are prepared in PBS0.5% (w/v)BSA/0.02% (w/v)Tween 20 and antibody(MOR08159) is added to each antigen concentration to reach a constantantibody concentration. 100 μl/well of each dilution mix is distributedin duplicates to a 96-well polypropylene MTP (Greiner). Assay bufferserved as negative control and a sample containing no antigen aspositive control (Bmax). The plate is sealed and incubated overnight. A96-well High Bind MTP (Meso Scale Discovery) is coated with 25 μl of 0.1μg/ml mouse ActRIIB-Fc diluted in PBS. Also this plate is sealed andincubated over night at 4° C. After the incubation the antigen-coatedHigh Bind MTP is washed with PBS/0.05% (w/v)Tween 20. Subsequently, theplate is blocked with PBS/5% (w/v) BSA. The washing steps are repeatedand 50 μl/well of the antibody-antigen preparation from thepolypropylene MTP is transferred into the antigen-coated High Bind MTP.The High Bind MTP is incubated for 25 min at room temperature. Afterthree additional washing steps, 25 μl of 1 μg/ml Sulfo-Tag-labeled goatanti-human-detection antibody (Meso Scale Discovery) diluted in assaybuffer is added to each well and incubated one hour at room temperature.After washing the plate, 50 μl of Read Buffer (Meso Scale Discovery) istransferred into each well. Electrochemiluminescence (ECL) signals aregenerated and detected by a Sector Imager 6000 reader (Meso ScaleDiscovery).

ECL values are plotted against the corresponding antigen concentrations.K_(D) is determined by fitting the plot with the fit model described byPiehler J, et al. (J Immunol Methods; 1997, 201(2): 189-206).

The reported K_(D) values and standard deviations are determined fromthe individual K_(D) values obtained from independent experiments.

From these experiments a mean value for the dissociation equilibriumconstant K_(D) of 1.73 (±0.31) pM is determined for human ActRIIB, whilea mean value for the dissociation equilibrium constant K_(D) of 434(±25) pM is determined for ActRIIA.

It will be understood that the disclosure has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the disclosure.

1. An anti-ActRIIB antibody or functional fragment thereof that binds toActRIIB between amino acids 19-134 of SEQ ID NO:
 181. 2. Theanti-ActRIIB antibody or functional fragment thereof according to claim1, wherein said antibody or functional fragment thereof is of the IgG₁isotype.
 3. The anti-ActRIIB antibody or functional fragment thereofaccording to claim 1, which has altered effector function throughmutation of the Fc region.
 4. An isolated polynucleotide sequenceencoding an antibody or functional fragment thereof according toclaim
 1. 5. A cloning or expression vector comprising one or moreisolated polynucleotide sequences according to claim
 4. 6. An isolatedhost cell comprising one or more vectors according to claim
 5. 7. Aprocess for the production of an anti-ActRIIB antibody or functionalfragment thereof that binds to ActRIIB between amino acids 19-134 of SEQID NO: 181, comprising culturing the host cell of claim 6 and isolatingsaid antibody or functional fragment thereof.
 8. A pharmaceuticalcomposition comprising an anti-ActRIIB antibody or functional fragmentthereof of claim
 1. 9. A pharmaceutical composition according to claim8, further comprising a pharmaceutically acceptable diluent or carrier.10. A pharmaceutical composition according to claim 8, furthercomprising one or more additional active agents.
 11. A pharmaceuticalcomposition according to claim 10, wherein said one or more additionalactive agents is selected from IGF-1, IGF-2, a variant of IGF-1 orIGF-2, an anti-myostatin antibody, a myostatin propeptide, a myostatindecoy protein that binds ActRIIB but does not activate it, a beta 2agonist, a Ghrelin agonist, a SARM, GH agonists/mimetics andfollistatin.
 12. A method of treatment of a musculoskeletal disease ordisorder in an individual in need thereof, comprising the step ofadministering an effective dose of an antibody or functional fragmentthereof according to claim
 1. 13. The method according to claim 12,wherein the musculoskeletal disease or disorder is muscle atrophy,myopathy, myotonia, a congential myopathy, nemalene myopathy,multi/minicore myopathy and myotubular (centronuclear) myopathy,mitochondrial myopathy, familial periodic paralysis, inflammatorymyopathy, metabolic myopathy, a glycogen or lipid storage disease,dermatomyositisis, polymyositis, inclusion body myositis, myositisossificans, rhabdomyolysis and myoglobinurias; a dystrophy, Duchenne,Becker, myotonic, fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal,scapulohumeral, limb girdle, Fukuyama, or a congenital musculardystrophy, or hereditary distal myopathy; osteoporosis; a bone fracture;short stature; dwarfism; prolonged bed rest; voluntary inactivity;and/or involuntary inactivity.
 14. The method according to claim 13,wherein the individual has been pre-treated with IGF-1, IGF-2 orvariants of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatinpropeptide, a myostatin decoy protein that binds ActRIIB but does notactivate it, a beta 2 agonist, a Ghrelin agonist, a SARM, GHagonists/mimetics or follistatin.
 15. The method according to claim 13,wherein the individual has previously been refractive to treatment withIGF-1, IGF-2 or variants of IGF-1 or IGF-2, an anti-myostatin antibody,a myostatin propeptide, a myostatin decoy protein that binds ActRIIB butdoes not activate it, a beta 2 agonist, a Ghrelin agonist, a SARM, GHagonists/mimetics or follistatin.
 16. The method according to claim 13,wherein the individual is elderly, has spent time in a zero gravityenvironment, or has undergone a period of inactivity.
 17. The methodaccording to claim 13, wherein the individual has a fracture to a limbor joint.
 18. The method according to claim 13, wherein the individualhas undergone or is about to undergo, hip or knee replacement surgery.